Anti-HIV domain antibodies and method of making and using same

ABSTRACT

The invention provides single domain antibodies and derivatives thereof that bind antigens of interest, which are stable, soluble, and do not tend to aggregate. The invention also provides methods for constructing a dAb library and methods for screening dAb libraries to identify the dAb of the invention. The invention also provide methods of treating or preventing conditions by antigen neutralization by administering the dAbs of the invention.

CROSS REFERENCES TO RELATED APPLICATIONS

The invention described herein is a 35 U.S.C. §371 U.S. national entryof International Application PCT/US2009/030351, having an internationalfiling date of Jan. 7, 2009, which claims priority to U.S. ProvisionalApplication Ser. No. 61/019,426, filed Jan. 7, 2008, the entire contentsof all of which applications are incorporated herein by reference.

All documents cited or referenced herein and all documents cited orreferenced in the herein cited documents, together with anymanufacturer's instructions, descriptions, product specifications, andproduct sheets for any products mentioned herein or in any documentincorporated by reference herein, are hereby incorporated by reference,and may be employed in the practice of the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to domain antibodies, includingderivatives, fusions and/or fragments thereof, having specificity forHIV and to their identification and manufacture. The invention furtherrelates to the use of the domain antibodies of the invention intreating, preventing, and/or diagnosing various conditions, andparticularly, HIV infections.

2. Background

The life-cycle of the human immunodeficiency virus (both HIV-1 andHIV-2) is well known. HIV primarily infects cells of the human immunesystem, such as helper T cells (specifically CD4+ T cells), macrophagesand dendritic cells. Entry to cells of the immune system is mediatedthrough interaction of the virion envelope glycoproteins (gp120 andgp41) with the receptor CD4 on the target cells. In addition, viralentry is modulated through at least two co-receptors known as CXCR4 andCCR5, which are members of the chemokine receptor family of proteins,and have been shown to function with CD4 as coreceptors for HIV-1isolates that are tropic for T-cell lines or macrophages, respectively(Feng et al., 1996, Science 272:872-876; Alkhatib et al., 1996, Science272:1955-1958; Deng et al., 1996, Nature 381:661-666; Dragic et al.,1996, Nature 381:667-673). Other molecules in this family including CCR3and CCR2b, also appear to function as cofactors for some HIV-1 isolates(Doranz et al., 1996, Cell 85:1149-1158; Berson et al., 1997, J. Virol.71: 1692-1696; Choe et al., 1996, Cell 85:1135-1148).

Current anti-HIV therapy includes the use of compounds which inhibitvarious aspects of the HIV life-cycle, including entry, fusion andreplication in a target cell. While these therapies, particularly whenused in combination with one another, are effective, they are frequentlyshort-lived in that the viral strains rapidly develop resistance to oneor more of the compounds used—a widespread and major problem in thecurrent approaches in treating HIV infections.

Antibodies represent yet another promising approach in the treatment ofHIV infections. Human monoclonal antibodies (mAbs) currently representan important and growing technology in the development of inhibitors,vaccines, diagnostic and research tools. In fact, 22 mAbs have beenapproved by the US Food and Drug Administration against various diseasein the past several decades for various disease indications, includingrheumatoid arthritis (Centacor's REMICADE and Abbott Laboratories'HUMIRA), non-Hodgkin's lymphoma (Genentech's RITUXAN and IDEC's ZEVALIN)and respiratory syncytial virus infection (Medimmune's SYNAGIS). Manyother antibody drug candidates are in the late stages of clinical trialsand, as such, antibodies are now well established as both highly potentand well tolerated therapeutics.

However, no mAbs have yet been approved for clinical use against HIV-1.A fundamental problem in the development of HIV-1-neutralizingantibodies is the virus's innate ability to escape human immunesurveillance during the long chronic infection. Several known mAbs,however, have been shown to exhibit potent and broad HIV-1 neutralizingactivity in vitro, and can prevent HIV-1 infection in animal models(reviewed in Burton, 2002, Ferrantelli et al., 2002, and Veazey et al.,2003). A recent clinical trial suggested that two of these broadly HIV-1neutralizing human mAbs, 2F5 and 2G12, lack side effects in humans(Armbruster et al., 2002; Stiegler et al., 2002). However, the potencyof 2F5 and 2G12 used in combination in this clinical trial wassignificantly lower than currently available treatments and relapsesoccurred (Stiegler et al., 2002). Further increase in the potency ofanti-HIV antibodies and/or new, more effective anti-HIV antibodies wouldbe a significant advancement in the art.

Another fundamental problem in the development of effective therapeuticantibodies against HIV is the problem of epitope accessibility. It hasbeen reported that some epitopes are sterically inaccessible to fullsize antibodies. For example, a study relating to HIV CD4-inducible(CD4i) epitopes by one of the present inventors has suggested that thesize of the CD4i-specific neutralizing antibodies inversely correlateswith neutralization efficiency and that perhaps antibody fragments mightbe more effective than whole antibodies in neutralizing the virus atsuch epitopes. See Labrijn et al., J. Virol., 2003. The study suggeststhat HIV's ability to evade the host's immune system may be linked inpart to its having found a way to sterically hinder full-sizedantibodies from accessing the CD4i epitopes. This study was limited,however, to exploring the effectiveness of scFv and Fab antibodyfragments.

In the late 1980s, domain antibodies were identified as the smallestknown antigen-binding fragments (Holt et al., 2003). Structurally,domain antibodies comprise the single chain variable heavy (VH) orvariable light (VL) polypeptides, and due to their single-chain nature,range in size of only 11 kDa to 15 kDa. Domain antibodies, however, havea number of acknowledged problems to overcome to be suitable aspotential therapeutics. Domain antibodies, particularly those derivedfrom human antibodies, suffer from poor stability and solubility, andhave a tendency to aggregate due to exposed regions of hydrophobicity inthe absence of the paired VH or VL.

New and effective domain antibodies which would overcome the problems inthe art, and an effective means of identifying and obtaining such domainantibodies, would be a valuable advance in the art. Such antibodiescould be the basis of new methods and approaches for treating and/orprophylaxis of a variety of infections and conditions, such as HIV orcancer, in particular, infections and conditions which are capable ofevading the immune system or therapeutic compounds and antibodiesbecause certain epitope targets are sterically restricted.

SUMMARY OF THE INVENTION

The present invention relates to single domain antibodies that overcomethe known problems in the art relating to domain antibodies and otheranti-HIV antibodies, particularly poor stability, solubility andeffectiveness. The domain antibodies of the invention show high affinityfor their target epitopes, are highly expressed, are stable, and arecapable of potent neutralization of a broad range of HIV isolates. Theinvention also relates to a novel VH framework identified by the presentinventors that can be used as the basis of a highly diverse dAb libraryfrom which the inventive domain antibodies having potent neutralizationactivity against a broad range of HIV isolates can be obtained. Thenovel VH framework of the invention unexpectedly showed a high degree ofcompatibility with a wide diversity of CDR sequences, displayed properlyfolded dAbs, and expressed dAbs at high levels. The present inventionalso relates to novel fusion proteins containing the domain antibodiesof the invention fused to agents (e.g., proteins) which function toenhance the stability of the dAbs of the invention and/or to enhance theeffectiveness of the domain antibodies against their targets. Inaddition, the present invention provides therapeutic methods employingthe domain antibodies and fusion proteins comprising the domainantibodies of the invention for treating and/or preventing HIVinfections. Methods for preparing and screening domain antibodylibraries are also provided herein.

In one embodiment, the present invention provides an isolated domainantibody or fragment thereof according to the amino acid sequence ofm36, or an amino acid molecule having at least 60%, 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% sequence identity with the amino acid sequence ofm36. In one aspect, the domain antibody or fragment can beimmunoconjugated to one or more cytotoxic agents, chemotherapeuticagents, natural or synthetic toxins, radioactive isotopes, or antiviralagents. In another aspect, the antiviral agent can be zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir,indinavir, amprenavir, lopinavir, ritonavir, adefovir, clevadine,entecavir, or pleconaril.

In another embodiment, the present invention provides an isolated domainantibody or fragment thereof comprising (a) the m0 framework amino acidsequence or an amino acid sequence having at least 60%, 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% sequence identity thereto and (b) the CDR3amino sequence of m36 or an amino acid sequence having at least 60%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. Inanother aspect, the domain antibody or fragment thereof can beimmunoconjugated to one or more cytotoxic agents, chemotherapeuticagents, natural or synthetic toxins, radioactive isotopes, or antiviralagents. In a further aspect, the antiviral agent can be zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir,indinavir, amprenavir, lopinavir, ritonavir, adefovir, clevadine,entecavir, or pleconaril.

In yet another embodiment, the present invention provides an isolateddomain antibody or fragment thereof comprising (a) the m0 frameworkamino acid sequence or an amino acid sequence having at least 60%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto and (b) atleast one of CDR1, CDR2 or CDR3 of m36 or an amino acid sequence havingat least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identitythereto. In one aspect, the domain antibody or fragment thereof can beimmunoconjugated to one or more cytotoxic agents, chemotherapeuticagents, natural or synthetic toxins, radioactive isotopes, or antiviralagents. In another aspect, the antiviral agent can be zidovudine,acyclovir, gangcyclovir, vidarabine, idoxuridine, trifluridine, andribavirin, as well as foscarnet, amantadine, rimantadine, saquinavir,indinavir, amprenavir, lopinavir, ritonavir, adefovir, clevadine,entecavir, or pleconaril.

In still another embodiment, the present invention relates to a domainantibody framework for the construction of a domain antibody library,said domain antibody framework comprising the framework sequence of m0or an amino acid sequence having at least 60%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity thereto.

In yet another embodiment, the present invention provides a fusionprotein comprising a domain antibody or fragment thereof and a fusionpartner, wherein the domain antibody or fragment thereof is the aminoacid sequence of m36, or an amino acid molecule having at least 60%,80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with theamino acid sequence of m36. In yet another embodiment, the presentinvention relates to a fusion protein comprising a domain antibody orfragment thereof and a fusion partner, wherein the domain antibody orfragment thereof comprises (a) the m0 framework amino acid sequence oran amino acid sequence having at least 60%, 80%, 85%, 90%, 95%, 96%,97%, 98% or 99% sequence identity thereto and (b) at least one of CDR1,CDR2 or CDR3 of m36 or an amino acid sequence having at least 60%, 80%,85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto.

In certain aspects, the fusion protein can be immunoconjugated to one ormore cytotoxic agents, chemotherapeutic agents, natural or synthetictoxins, radioactive isotopes, or antiviral agents. The antiviral agentcan be zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine,trifluridine, and ribavirin, as well as foscarnet, amantadine,rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir,adefovir, clevadine, entecavir, or pleconaril.

In yet another aspect, the fusion partner of the fusion proteins of theinvention can be fused to the m36 or fragment thereof via a linker, suchas, for example, a human IgG hinge region.

In certain other aspects, the fusion partner of the fusion proteins ofthe invention increases the stability of m36 or fragment thereof ascompared to the m36 or fragment thereof alone. In certain other aspects,the fusion partner of the fusion proteins of the invention induces aCD4i epitope on Env thereby synergistically increases the effectivenessof the domain antibodies of the invention.

In one aspect, the fusion partner of the fusion proteins of theinvention can be serum albumin-binding protein.

In certain other aspects, the fusion partner of the fusion proteins ofthe invention can be CD4 or a fragment or mimic thereof.

The fusion protein of claim 21, wherein the fusion partner is fused tothe domain antibody or fragment thereof via a linker. In a furtheraspect, the linker can be an IgG hinge region.

In still another embodiment, the present invention provides apharmaceutical composition comprising a therapeutically effective amountof a domain antibody or a fusion protein in accordance with theinvention and a pharmaceutically acceptable salt.

In another embodiment, the invention relates to a method of treatingand/or preventing an HIV infection in a patient in need thereofcomprising, administering a therapeutically effective amount of (a) adomain antibody of the invention (b) a fusion protein of the invention,or (c) a pharmaceutical composition comprising a domain antibody or afusion protein of the invention. In one aspect, the treatment method canfurther include co-administering a cytokine, anti-angiogenic agent,immunotherapeutic agent, anti-cancer agent, anti-bacterial agent, oranti-viral agent. In still another aspect, the treatment method furthercomprises co-administering a therapeutically effective amount of solubleCD4 (sCD4) or functional fragment or mimic thereof.

In yet another embodiment, the present invention provides a domainantibody library comprising a plurality of unique clones wherein eachunique clone comprises the framework sequence of m0 or an amino acidsequence having at least 60%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%sequence identity thereto. In one aspect, the domain antibody librarycomprises at least 10⁸ or 10⁹ or 10¹⁰ unique clones.

In still another embodiment, the present invention provides a method ofconstructing a dAb library comprising the steps of (a) obtaining anisolated VH framework nucleotide sequence and (b) introducing a CDR1,CDR2 and CDR3 repertoire into the isolated VH framework nucleotidesequence, thereby forming a dAb library. In one aspect, the step ofintroducing the CDR1, CDR2 and CDR3 repertoire is achieved by graftreplacement of the corresponding CDR sequences of the VH framework withthe CDR1, CDR2 and CDR3 repertoire. In another aspect, the CDR1, CDR2and CDR3 repertoire sequences can be obtained by PCR amplification usingthe primers of Table 1 and a non-immunized or immunized human antibodylibrary template. In still another aspect, the graft replacement can beachieved by extension reactions between the PCR CDR products and the VHframework. In another aspect one, two or all CDRs could be mutagenized.The dAb library of the invention can be a phage-display library.

In still another embodiment, the present invention provides a method ofidentifying a dAb that binds to an antigen of interest, comprisingpanning the dAb phage-display library of the invention with an antigenof interest. In one aspect, the antigen of interest is HIV-1 CD4iantigen. In another aspect, the panning can be sequential panning usingat least two antigens. The at least two antigens can be two HIV-1 CD4iantigens from different HIV-1 isolates.

In still another embodiment, the present invention provides a method fortreating or preventing an HIV infection comprising administering in atherapeutically effective amount a composition comprising a dAb thatbinds to HIV-1 CD4i with a dissociation constant (K_(d)) of about 1 nMto about 500 nM.

These and other embodiments are disclosed or are obvious from andencompassed by, the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the invention solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings.

FIG. 1 is a simplified depiction of the general structure of the HIVenvelope glycoprotein (Env) bound to CD4 and a coreceptor at the surfaceof a target cell. The HIV Env complex is embedded in the HIV envelopeand consists of three (3) molecules of gp120 and a stem consisting ofthree (3) molecules gp41 of which only one is shown. The coreceptor caninclude chemokine receptors, CCR5 or CXCR4.

FIG. 2 depicts a general overview of a phage display antigen panningprocess that can be used to identify the antibodies of the invention.

FIG. 3 depicts in ribbon form the general structure of a human IgGmolecule, with emphasis on the VH region, which may form a domainantibody of the invention.

FIG. 4 is a photograph of an SDS-PAGE gel showing the R3H1Fab and its VHdomain, m0, of the invention. The VH domain, m0, was identified bypanning a human naïve antibody library, which had been recentlyconstructed according to the procedure and primers described in (deHaard H J et al. 1999. J. Biol. Chem. 274:18218-30) but with one-stepoverlapping PCR cloning (Zhu Z and Dimitrov D S, in press, Therapeuticantibodies, A. Dimitrov, Ed., Methods in Molecular Biology, HumanaPress), with an HIV-1 antigen, recombinant soluble Env with truncatedtransmembrane portion and cytoplasmic tail—gp140. The R3H1Fab wasidentified with a stop codon in the light chain, after which its readingframe was corrected. The VH domain of R3H1 was sub-cloned into aphagemid vector, expressed and purified.

FIG. 5 depicts the m0 VH framework. FIG. 5A compares the m0 gene and thededuced amino acid sequence to human VH germline sequences from IMGT(ImMunoGeneTics) database(http://imgt.cines.fr/textes/IMGTrepertoire/Proteins/protein/human/IGH/IGHV/Hu_IGHVallgenes.html).The alignments to the known human VH germline sequences indicatedVH3-23*04 as having the closest sequence identity with m0 although thereare still significant differences. The figure displays the m0 amino acidsequence on the top row, followed below by its corresponding nucleotidesequence, and followed below the aligned VH3-23*04 sequences showingonly those amino acid residues and nucleotide residues differing fromthe m0 sequences. The CDR (complementary determining region) and FR(framework) regions are shown according to IMGT numbering system. Theitalicized regions flanking the CDR2 and CDR3 regions at their 5′ and 3′ends correspond to the target sites for the primers used herein toamplify CDR2 and CDR3 segments from other sources in accordance with theinvention, e.g. as described in Example 2. FIG. 5B provides separateamino acid and nucleotide sequences for m0, indicating the locations ofthe CDR sequences.

FIG. 6 illustrates a process of constructing the VH library, whichcomprises the m0 framework and a diversity of CDR sequences fromdifferent sources. The VH library was constructed in three steps. In thefirst step, PCRs were performed to amplify CDR2 and CDR3 gene segmentsfrom various antibody libraries (plasmid DNA containing human antibodygene fragments). CDR2 amplification products from different librarieswere pooled. Similarly, CDR3 amplification products generated fromdifferent libraries were pooled together. The CDR1 segment was preparedby PCR amplification from a m0 template using the degenerate primer H1Rcovering the entire length of CDR1 to generate mutations at fourpositions to A, D, S or Y. The FR3 segment was also obtained from m0 forassembly of entire VHs. In the second step, overlapping PCRs wereperformed to join CDR1s to CDR2s and FR3 to CDR3s, respectively. In thethird step, entire VHs were assembled by overlapping PCR of the productsof the second step. The products were cloned into a phagemid vector anda library was obtained by performing electroporations as described inthe Examples.

FIG. 7 is a photograph of an SDS-PAGE gel showing soluble VHs selectedagainst HIV-1 antigens. Twelve positive clones (c3, c6, d1, d7, b4, c11,d10, b3, b5 (m36), b7, g6 and e11) were selected after three rounds ofsequential panning of library with HIV-1 antigens gp120-CD4 and gp140,expressed and purified by immobilized metal ion affinity chromatography(IMAC) using Ni-NTA resin.

FIG. 8 illustrates the sequence diversity analysis of the VH domainlibrary of the invention, and as described in Example 2. To evaluate thesequence diversity of the library, clones were randomly selected andsequenced. Regarding the diversity of CDR1 sequences, FIG. 8A shows thefrequency of A/D/S/Y usage in each mutated position. Regarding thediversity of CDR2, FIG. 8B provides two bar graphs that show the numberof VH germline subgroups of 1-7 as compared to the frequency of theappearance of the corresponding CDR2 in the library. FIG. 8C is a bargraph showing the frequency of mutations in CDR2. Regarding CDR3sequence diversity, FIG. 8D provides bar graphs comparing the frequencyof CDR3 lengths in vivo against the frequency of library clones havingthe same lengths.

FIG. 9 shows the diversity in the lengths of the CDR3s of the differenthuman VH germlines genes.

FIG. 10 shows the frequency of A/D/S/Y residues at positions 27, 28, 29,30, 31 and 32 in CDR1 before selection against Protein A versus afterselection against Protein A in accordance with Example 2. Selectionagainst Protein A was carried out, as explained in Example 2, toevaluate the extent to which correct folding occurred in the VH libraryof phagemid clones.

FIG. 11 shows SDS-PAGE gels of three sets of clones grouped in A, B andC gels. For each gel, the clone number, the origin of the CDR2, thelength in amino acids of CDR3, and the yield in milligrams are indicatedfor each set of clones. See Example 2 for further description. Thefigure shows the expression profile before (11A and B) 300 and after(11C) panning with antigens.

FIG. 12 shows the results of size exclusion chromatography analysis tomeasure the oligomerization of select isolated VHs, c6, d7, b5 (m36), b7and d10. See Example 2 for further description.

FIG. 13 shows the results of size exclusion chromatography analysis tomeasure the oligomerization of m36. Purified m36 in PBS was subjected tosize exclusion chromatography with Superdex75 column calibrated withprotein molecular mass standard shown by the arrows.

FIG. 14 shows the binding specificity of m36. Binding of m36 was testedby ELISA using Corning high-binding 96-well plates coated with 1 μg/mlof antigens. Bound m36 were detected by adding 1:5000 dilutedHRP-conjugated anti-FLAG antibody. The assay was developed at 37° C.with ABST substrate and monitored at 405 nm as described.

FIG. 15 shows that the binding of m36 to gp120 is induced by the gp120interaction with CD4. gp120_(Bal)-CD4 was coated on Corning high-binding96-well plates. M36 with consensus concentration was mixed with either aknown CD4i antibody (m16) or CD4bs antibody (m14) (negative control) inIgG format serially diluted, and added to 96-well plates coated withantigen. Bound m36 were detected by 1:5000 diluted HRP-conjugatedanti-FLAG antibody. The assay was developed at 37° C. with ABSTsubstrate and monitored at 405 nm. See FIG. 27 for complementary datashowing the specific binding of m36 to gp120_(Bal)-CD4 but not togp120_(Bal) alone.

FIG. 16 shows the cross-reactivity of m36. GXC gp140 from an isolate ofclade C was coated in the presence or absence of CD4. Serially dilutedm36 was added and detected by HRP-conjugated anti-FLAG antibody. Theassay was developed at 37° C. with ABST substrate and monitored at 405nm.

FIG. 17 shows the results of testing the IC₅₀ (the minimum inhibitoryconcentration needed to inhibit or neutralize 50% of the total virus),IC₉₀ (the minimum inhibitory concentration needed to inhibit orneutralize 90% of the total virus) and % In (percentage inhibition forthe highest concentration of antibody—m36: 10 μg/ml; scFv m9: 20 μg/ml;c34: 4 μg/ml) of m36 antibody, scFv m9 antibody, and c34 antibodyagainst 11 different HIV-1 isolates from a total of 5 clades, A, B, C, Dand E.

FIG. 18 shows the amino acid and nucleotide sequences for m36. The CDR1,CDR2 and CDR3 sequences are indicated. Also presented are the amino acidand nucleotide sequences of the human germline gene VH3-48*03. Onlythose amino acid and nucleotide residues that differ from the m36sequences are indicated in the figure.

FIG. 19 provides the nucleotide and amino acid sequences of m36.

FIG. 20 provides a schematic representations of the m0 master frameworkand the cloning region of phagemid vector. (A) m0 master framework isanalyzed using IMGT/V-QUEST tool provided by IMGT immunoglobulindatabase(http://imgt.cines.fr/IMGT_vquest/vquest?livret=0&Option=humanIg). TheFRs and CDRs regions of the master gene are indicated according to thedatabase. (B) Brief description of the cloning region of the phagemidvector phagemid vector. SfiI restriction sites are frequently used forcloning of genes. Hexahistidine tag and HA tag are included forpurification and detection of protein products.

FIG. 21 shows the preparation of cDNA by reverse transcription of totalRNA from human peripheral blood mononuclear cells. Total RNA wasextracted from human mononuclear cells with RNeasy Mini Kit (Qiagen,Cat. #74104) as described below in Example 2. Using a SuperScript IIIFirst-Strand Synthesis System (Invitrogen, Cat. #18080-051) containingoligo (dT)₂₀ primers and random hexamers, total RNA was then reversetranscribed into cDNA. The cDNA products were separated on a 0.8% (w/v)agarose gel. The cDNA 1 and cDNA 2 are from reactions using oligo (dT)₂₀primers and random hexamers, respectively. Two molecular weight DNA massmarkers, Marker 1 (Invitrogen Cat. #15628-019) and Marker 2 (InvitrogenCat. #10787-018) were included.

FIG. 22 demonstrates the results obtained from PCR amplification of CDR2and CDR3 repertoires from cDNA. (A) Eight recombinations of primers wereused for CDR2s amplification as described below in Example 2. Theproducts of the first five recombinations (H2-F1/H2-R1, H2-F1/H2-R2,H2-F1/H2-R5, H2-F2/H2-R3, H2-F3/H2-R1, and H2-F3/H2-R2) were shown onlane 1 to lane 5, respectively. (B) Three recombinations of primers wereused for CDR3 amplification. The products of the first tworecombinations (H3-F1/H3R and H3-F2/H3R) were shown on lane 1 and 2,respectively. The correct-sized bands were indicated by arrows.

FIG. 23 depicts the construction of a human antibody VH library. Astable VH (m0) was used as a scaffold for grafting CDR2s and CDR3s fromfive human antibody Fab libraries. CDR1 residues #27, 29, 31 and 32(IMGT numbering system) were randomized to A, D, S or Y. The numbersdenote the positions of the amino acid residues corresponding to therespective regions of the antibody VH gene where the CDRs were grafted;the # denotes the positions of the CDR1 randomization. The SfiI denotesthe restriction enzyme sites used for cloning.

FIG. 24 shows an alignment of CD4i antibody VH sequences. The amino acidsequences of 12 known CD4i antibody VHs were obtained from a previousreport (Huang et al. (2004), Proc. Natl. Acad. Sci. USA, 101:2706-2711,the contents of which are incorporated herein by reference). A multiplesequence alignment of these antibodies in addition to m36 is shown withCDRs indicated. Sequences are ordered based on the CDR3 lengths; IMGTnumbering system is used. The acidic CDR residues are underlined withred. The gene usage with the FR and CDR regions and their CDR3 lengthsare shown on the right.

FIG. 25 provides a table showing pseudotyped virus neutralization by m36and its fusion proteins, as described below in Example 3. The tableprovides the antibody concentration (nM) resulting in 50% inhibition ofvirus infection (IC₅₀).

FIG. 26 shows potent m36-mediated neutralization of viruses pseudotypedwith Envs of HIV-1 primary isolates. (A) Dose-dependent inhibition ofBal by m36, scFv and C34, respectively. (B) Percentage inhibition of apanel of viruses by m36, scFv m9 and C34 at 667 nM, respectively.

FIG. 27 show that the binding of m36 to gp120 is induced by the gp120interaction with CD4. The graph shows the specific binding of m36 togp120_(Bal)-CD4 but not to gp120_(Bal) alone. See FIG. 15 forcomplementary data showing the competition of m36 with a CD4i antibody(IgG m16) for binding to gp120_(Bal)-CD4.

FIG. 28 depicts the design of m36 fusion proteins described in Example3. Schematic representation of m36 fused with SAbp, human IgG1 CH3domain or Fc. The names of the constructs and their molecular weightsare also shown. The sequences of the SAbp and the linkers used to joinm36 with Fc by human IgG1 and IgG3 hinge, and camel IgG2 hinge areQRHPEDICLPRWGCLWGDDD (SEQ ID NO: 1), DKTHTCPPCP (SEQ ID NO: 2),EPKIPQPQPKPQPQPQPQPKPQPKPEPECTCPKCP (SEQ ID NO: 3), andELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCP (SEQ ID NO: 4), respectively.

FIG. 29 depicts the cloning of m36 and its fusion proteins of FIG. 28.Schematic representation of the m36 gene cassettes, and its fusionproteins (A) and plasmids used for cloning (B).

FIG. 30 shows SDS-PAGE of m36 and its fusion proteins under reducing (−)and non-reducing (+) conditions.

FIG. 31 demonstrates that m36 and its fusion proteins have similarbinding activities. (A) Binding of m36, m36SAbp, m36CH3 and m36b0Fc togp120_(Bal)-CD4 and gp120_(Bal), respectively. (B) Binding of m36h1Fc,m36c2Fc and m36h3Fc to gp120_(Bal)-CD4 and gp120_(Bal), respectively.

FIG. 32 provides a table showing pseudotyped virus neutralization by m36and its fusion proteins, as described below in Example 3. The tableprovides the antibody concentration (nM) resulting in 90% inhibition ofvirus infection (IC₉₀).

FIG. 33 demonstrates that pre-triggering (sensitization) of virus bysCD4 dramatically increases neutralization by large molecules fused withm36. Viruses were pre-inoculated with different concentrations ofantibodies and/or sCD4 at 8 nM for 1 h at 37° C., and then the mixturewas added to 1.5×10⁴ HOS-CD4-CCR5 cells grown in each well of 96-wellplates. Luminesence was measured 48 h post infection and percentageinhibition was calculated as described in Example 3.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have developed new and useful single domainantibodies that overcome the known problems in the art relating todomain antibodies, particularly those problems associated with domainantibodies derived from human antibodies. Unlike the domain antibodiesof the art, the antibodies of the invention are stable, highly soluble,and do not tend to form aggregates or polymerization products insolution. In addition, the domain antibodies of the invention have highaffinity for their target epitopes and are highly expressed. Theadvantageous features of the inventive antibodies of the invention stemat least in part to the novel VH framework identified by the presentinventors that is used as the basis of a highly diverse dAb library fromwhich the inventive antibodies can be obtained. The novel VH frameworkof the invention unexpectedly and surprisingly showed a high degree ofcompatibility with a wide diversity of CDR sequences, maintains properfolding, expresses dAbs at high levels and which are highly soluble.

It is to be understood that present invention as described herein is notto be limited to the particular details set forth herein regarding anyaspect of the present invention, including, the anti-HIV domainantibodies and variants and/or fragments thereof, VH framework, methodof preparing diverse dAb library, methods of treatment, protocols, celllines, animal species or genera, constructs, immunoconjugates andreagents described and, as such, may vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by one of ordinary skill in the artto which this invention belongs.

As used herein, the term “antibody” refers to immunoglobulin molecules(e.g., any type, including IgG, IgE, IgM, IgD, IgA and IgY, and/or anyclass, including, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) isolated fromnature or prepared by recombinant means or chemically synthesized. Theterms “antibody” and “immunoglobulin” can be used synonymouslythroughout the specification, unless indicated otherwise.

As used herein, the term “antibody fragment” refers to a portion of awhole antibody which retains the ability to exhibit antigen bindingactivity. Examples include, but are not limited to, Fv,disulphide-linked Fv, single-chain Fv, Fab, variable heavy region(V_(H)), variable light region (V_(L)), and fragments of any of theabove antibody fragments which retain the ability to exhibit antigenbinding activity, e.g., a fragment of the variable heavy region V_(H)retains its ability to bind its antigen.

As used herein, the term “antibody framework” is intended to mean theportion of an antibody variable domain which serves as a scaffold forthe antigen binding loops of the variable domain, i.e., the CDRsequences, which is the same definition proffered by Kabat et al. 1991,which is incorporated herein by reference).

As used herein, the term “complementarity determining regions” (CDRs),or synonymously, antibody CDR, refers to the amino acid segments of anantibody that function as the antigen binding loops, as defined by Kabatet al. (1991). Each of the two variable domains of an antibody Fvfragment each contain three CDRs. The CDRs for the antigen bindingloops, or synonymously, the “antigenic binding site”.

As used herein, the term “domain antibody” (dAb) refers to an antibodywhose complementary determining regions (CDRs) are part of a singledomain polypeptide. Examples include, but are not limited to, variableheavy region (V_(H)) or fragment thereof, variable light region (V_(L))or fragment thereof, heavy chain antibody (i.e. antibody devoid of lightchain), single domain antibodies derived or engineered from conventional4-chain antibodies, engineered antibodies and single domain scaffolds,wherein the scaffolds can be derived from any natural or syntheticsource of antibody. The CDRs can be from any natural or syntheticsource, and can be modified by any known or suitable means, e.g.site-directed mutagenesis.

As used herein, the term “framework region” refers to the nucleic acidsequence regions of an antibody gene that encode the structural elementsof the molecule. In a domain antibody, e.g. the VH or VL of a human IgG,the framework region represents the sequences surrounding each of thethree CDR sequences of the fragment polypeptides.

As used herein, the term “library” refers to a collection of nucleicacid sequences, wherein the individual nucleic acid molecules arecarried or contained in a suitable vector, e.g. a DNA vector, expressionvector, phagemid vector.

As used herein, the term “naive library” refers to a collection ofnucleic acid sequences encoding the naturally occurring V_(H) or V_(L)repertoire from a non-immunized source.

As used herein, the term “repertoire” refers to the genetic diversity ofa collection of molecules, e.g. a collection of CDR sequences.

As used herein, the terms “biological sample” or “patient sample” asused herein, refers to a sample obtained from an organism or fromcomponents (e.g., cells) of an organism. The sample may be of anybiological tissue or fluid. The sample may be a clinical sample which isa sample derived from a patient. Such samples include, but are notlimited to, sputum, blood, serum, plasma, blood cells (e.g., whitecells), tissue samples, biopsy samples, urine, peritoneal fluid, andpleural fluid, saliva, semen, breast exudate, cerebrospinal fluid,tears, mucous, lymph, cytosols, ascites, amniotic fluid, bladder washes,and bronchioalveolar lavages or cells therefrom, among other body fluidsamples. The patient samples may be fresh or frozen, and may be treated,e.g. with heparin, citrate, or EDTA. Biological samples may also includesections of tissues such as frozen sections taken for histologicalpurposes.

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural reference unless the context clearlydictates otherwise. Thus, for example, reference to “a gene” is areference to one or more genes and includes equivalents thereof known tothose skilled in the art, and so forth.

As used in this invention, the term “epitope” means any antigenicdeterminant on an antigen, e.g. a gp120 protein, to which an antibodybinds through an antigenic binding site. Determinants or antigenicdeterminants on an antigen usually consist of chemically active surfacegroupings of molecules such as amino acids or sugar side chains andusually have specific three dimensional structural characteristics, aswell as specific charge characteristics. In one embodiment of theinvention, the HIV-1 epitope is the CD4-inducible epitope (“CD4i”),which becomes exposed on gp120 only after gp120 binds to the CD4receptor.

As used herein, the term antibody that “specifically binds to” or is“specific for” a particular polypeptide or an epitope on a particularpolypeptide is one that binds to that particular polypeptide or epitopeon a particular polypeptide without substantially binding to any otherpolypeptide or polypeptide epitope. Alternatively, an antibody thatspecifically binds to an antigen, in accordance with this invention,refers to the binding of an antigen by an antibody or fragment thereofwith a dissociation constant (K_(d)) of 1 μM or lower, as measured bysurface plasmon resonance analysis using, for example, a BIACORE®surface plasmon resonance system and BIACORE® kinetic evaluationsoftware (eg. version 2.1). The affinity or dissociation constant(K_(d)) for a specific binding interaction is preferably about 500 nM orlower, more preferably about 300 nM or lower and preferably at least 300nM to 50 pM, 200 nM to 50 pM, and more preferably at least 100 nM to 50pM, 75 nM to 50 pM, 10 nM to 50 pM.

As used herein, the term “fusion protein” refers to two or morepolypeptides coupled together which are not naturally found in a coupledarrangement. This can include translational fusions of two polypeptides,e.g., an antibody of the invention translationally fused via recombinantmeans to a fusion partner, such as an adduct molecule to enhance thestability of the antibody. Fusion proteins can also include proteins,e.g., an antibody of the invention, which have been physically coupledto another polypeptide. The two polypeptides in either case can bejoined via a linker molecule.

As use herein, the term “fusion partner” refers to each of thepolypeptides of a fusion protein.

As used herein, the term “linker” refers to a flexible molecularconnection between two or more proteins, e.g., the linker between twom36 molecules. The molecular connection can be obtained from anysuitable natural or synthetic source. The linker can be obtained, forexample, from an antibody hinge region, e.g., IgG hinge region or fromanother natural or synthetic polypeptide source. The linker can beencoded, as in translational fusions. The linker can also be used tojoin or couple two already existing proteins.

As used herein, the term “mimic” refers to a second molecule, compoundor substance which has the same or similar function or characteristicsas a first molecule, compound or substance, while at the same timehaving a different structure.

VH Framework

In one embodiment, the present invention provides a novel andadvantageous VH framework, which the present inventors have used as thestarting point for generating a highly diversified phage-displayeddomain antibody library which, in turn, can be used as convenient sourceof domain antibodies which are unexpectedly highly soluble and stableand possess good folding fidelity (despite a wide diversity of CDRmolecules) and high affinity for their cognate antigenic targets.

In a preferred embodiment of the invention, the present inventionprovides a VH framework having the amino acid and nucleotide sequence ofFIG. 5 (indicated as m0, herein as “the m0 framework”). The constructionof the m0 framework is described in Example 2 herein. Essentially, them0 framework is the VH region of the Fab antibody, R3H1, which wasidentified by screening a large non-immune human Fab library (containing˜1.5×10¹⁰ members) derived from the lymph nodes, spleen and peripheralblood lymphocytes of 50 human donors. As shown in Example 2, the m0framework was found to have high levels of expression and highsolubility. This completely natural VH domain antibody, belonging to theVH3 germline family, was then used as a framework to construct a largehuman VH domain library (with ˜2.5×10¹⁰ members) by grafting in adiverse repertoire of CDRs and/or mutating existing framework CDRs.

In another embodiment, the present invention provides a VH frameworkderived from m0, wherein the m0 contains certain advantageous amino acidmodifications to enhance the properties of the framework antibody,including its solubility, stability, and lack of tendency to aggregate.Recombinant DNA techniques for modifying and/or changing the nucleicacid sequence and/or amino acid sequence of antibodies, including m0,are well known to those having ordinary skill in the art. For example,techniques for introducing genetic changes include site-directedmutagenesis, random mutagenesis, insertions, deletions, and PCRmutagenesis methods. All of these techniques are well known to thoseskilled in the art. See Ausubel et al., Current Protocols in MolecularBiology, John Wiley & Sons, New York, 2000, incorporated herein byreference.

Accordingly, the present invention relates to VH framework polypeptidesthat are at least 60%, preferably at least 80% identical, morepreferably at least 90%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of the m0 framework.

The invention also relates to the nucleic acid encoding the VH frameworkm0 as shown in FIG. 5, or a VH framework nucleic acid sequence that isat least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotidesequence of the m0 framework.

As used herein, the terms percent (%) sequence identity or percent (%)homology are used synonymously as a measure of the similarity of two ormore amino acid sequences, or alternatively, between two or morenucleotide sequences. Methods for determining percent (%) sequenceidentity or percent (%) homology are well known in the art.

For the purposes of the present invention, percent (%) sequence identityor homology can be determined by comparing the sequences when aligned soas to maximize overlap and identity while minimizing sequence gaps. Inparticular, sequence identity may be determined using any of a number ofmathematical algorithms. A nonlimiting example of a mathematicalalgorithm used for comparison of two sequences is the algorithm ofKarlin & Altschul, Proc. Natl. Acad. Sci. USA 1990; 87: 2264-2268,modified as in Karlin & Altschul, Proc. Natl. Acad. Sci. USA 1993; 90:5873-5877.

Another example of a mathematical algorithm used for comparison ofsequences is the algorithm of Myers & Miller, CABIOS 1988; 4: pp 11-17.Such an algorithm is incorporated into the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used. Yet another useful algorithm for identifying regions oflocal sequence similarity and alignment is the FASTA algorithm asdescribed in Pearson & Lipman, Proc. Natl. Acad. Sci. USA 1988; 85:2444-2448. Advantageous for use according to the present invention isthe WU-BLAST (Washington University BLAST) version 2.0 software. Thisprogram is based on WU-BLAST version 1.4, which in turn is based on thepublic domain NCBI-BLAST version 1.4 (Altschul & Gish, 1996, Localalignment statistics, Doolittle ed., Methods in Enzymology 266: 460-480;Altschul et al., Journal of Molecular Biology 1990; 215: 403-410; Gish &States, 1993; Nature Genetics 3: 266-272; Karlin & Altschul, 1993; Proc.Natl. Acad. Sci. USA 90: 5873-5877; all of which are incorporated byreference herein).

In general, comparison of amino acid sequences is accomplished byaligning an amino acid sequence of a polypeptide of a known structurewith the amino acid sequence of a the polypeptide of unknown structure.Amino acids in the sequences are then compared and groups of amino acidsthat are homologous are grouped together. This method detects conservedregions of the polypeptides and accounts for amino acid insertions anddeletions. Homology between amino acid sequences can be determined byusing commercially available algorithms (see also the description ofhomology above). In addition to those otherwise mentioned herein,mention is made too of the programs BLAST, gapped BLAST, BLASTN, BLASTP,and PSI-BLAST, provided by the National Center for BiotechnologyInformation. These programs are widely used in the art for this purposeand can align homologous regions of two amino acid sequences.

In all search programs in the suite the gapped alignment routines areintegral to the database search itself. Gapping can be turned off ifdesired. The default penalty (Q) for a gap of length one is Q=9 forproteins and BLASTP, and Q=10 for BLASTN, but may be changed to anyinteger. The default per-residue penalty for extending a gap (R) is R=2for proteins and BLASTP, and R=10 for BLASTN, but may be changed to anyinteger. Any combination of values for Q and R can be used in order toalign sequences so as to maximize overlap and identity while minimizingsequence gaps. The default amino acid comparison matrix is BLOSUM62, butother amino acid comparison matrices such as PAM can be utilized.

Alternatively or additionally, the term “homology” or “identity”, forinstance, with respect to a nucleotide or amino acid sequence, canindicate a quantitative measure of homology between two sequences. Thepercent sequence homology can be calculated as(N_(ref)−N_(dif))*100/−N_(ref), wherein N_(dif) is the total number ofnon-identical residues in the two sequences when aligned and whereinN_(ref) is the number of residues in one of the sequences. Hence, theDNA sequence AGTCAGTC will have a sequence identity of 75% with thesequence AATCAATC (N_(ref)=8; N_(dif)=2).

Alternatively or additionally, “homology” or “identity” with respect tosequences can refer to the number of positions with identicalnucleotides or amino acids divided by the number of nucleotides or aminoacids in the shorter of the two sequences wherein alignment of the twosequences can be determined in accordance with the Wilbur and Lipmanalgorithm (Wilbur & Lipman, Proc Natl Acad Sci USA 1983; 80:726,incorporated herein by reference), for instance, using a window size of20 nucleotides, a word length of 4 nucleotides, and a gap penalty of 4,and computer-assisted analysis and interpretation of the sequence dataincluding alignment can be conveniently performed using commerciallyavailable programs (e.g., Intelligenetics™ Suite, Intelligenetics Inc.CA). When RNA sequences are said to be similar, or have a degree ofsequence identity or homology with DNA sequences, thymidine (T) in theDNA sequence is considered equal to uracil (U) in the RNA sequence.Thus, RNA sequences are within the scope of the invention and can bederived from DNA sequences, by thymidine (T) in the DNA sequencebeing-considered equal to uracil (U) in RNA sequences.

And, without undue experimentation, the skilled artisan can consult withmany other programs or references for determining percent homology.

The modifications to the amino acid sequence of the m0 framework to formvariants that are at least 60%, preferably at least 80% identical, morepreferably at least 90%, 95%, 96%, 97%, 98% or 99% identical to theamino acid sequence of the m0 framework, can be made using conservativeamino acid substitutions. A conservative substitution is one in whichthe substituted amino acid has similar structural or chemical propertieswith the corresponding amino acid in the reference sequence. By way ofexample, conservative amino acid substitutions involve substitution ofone aliphatic or hydrophobic amino acid, e.g. alanine, valine, leucineand isoleucine, with another; substitution of one hydroxyl-containingamino acid, e.g. serine and threonine, with another; substitution of oneacidic residue, e.g. glutamic acid or aspartic acid, with another;replacement of one amide-containing residue, e.g. asparagine andglutamine, with another; replacement of one aromatic residue, e.g.phenylalanine and tyrosine, with another; replacement of one basicresidue, e.g. lysine, arginine and histidine, with another; andreplacement of one small amino acid, e.g., alanine, serine, threonine,methionine, and glycine, with another.

Domain Antibody Library Construction and Screening Methods

In yet another embodiment, the present invention provides a domainantibody library prepared by introducing CDR diversity into the m0framework (or derivative thereof having various amino acid substitutionsas described above). Any suitable method for introducing CDR diversityis contemplated, including mutagenesis of one or more of the existing m0CDR1, CDR2 or CDR3 sequences and the grafting in or replacement of oneor more of the existing m0 CDR1, CDR2 or CDR3 sequences with a diverserepertoire of synthetically-prepared or naturally-obtained CDRsequences. The naturally-obtained CDR sequences can comprise a naiverepertoire (not exposed to antigen) or an immunized repertoire (exposedto antigen).

The CDR sequences may be obtained from several sources, for example,databases such as the National Centre for Biotechnology Informationprotein and nucleotide databases www.ncbi.nlm.nih.gov, The KabatDatabase of Sequences of Proteins of Immunological Interestwww.kabatdatabase.com, or the IMGT database www.imgt.cines.fr.Alternatively, the CDR regions can be predicted from the VH and VLdomain repertoire (see for example Kabat E A and Wu T T Attempts tolocate complementarity determining residues in the variable positions oflight and heavy chains. Ann. NY Acad. Sci. 190:382-393 (1971)). The CDRsequence may be a genomic DNA or a cDNA.

PCR amplification using primers that amplify the CDR1, CDR2 and CDR3sequences, which may include framework sequence at the 5′ and 3′ ends ofthe CDR sequences, can be used to obtain the CDR repertoire for graftinginto the framework sequence of the invention. Such PCR methods will beknown to those of ordinary skill in the art. CDR sequences can also beobtained by any known nucleotide sequence synthesis methods, or byobtaining DNA fragments (e.g. restriction fragments) carrying the CDRsequences of interest. Any suitable method for obtaining the CDRsequences of the invention for use in grafting into the frameworksequence is contemplated.

In a particular embodiment, diversity can be introduced into the m0framework by grafting in a natural repertoire of one or more human CDR1,CDR2 or CDR3 sequences, obtained from known and available human antibodylibraries, while synthetically modifying at least one existing CDRsequence of the m0 framework. Alternatively, diversity can be introducedby either only grafting in a CDR1, CDR2 and CDR3 repertoire, or bysynthetically modifying each existing CDR sequence of the framework,without simultaneous grafting. Human antibody libraries can be obtainedfrom any suitable source (e.g. commercial, university laboratories,public repositories of antibody libraries) and can include, for example,both immunized and non-immunized type libraries. As used herein, anon-immunized antibody library is one in which the patient(s) from whichthe antibodies were derived has not been exposed to the antigen againstwhich the domain antibody library will ultimately be screened. Animmunized antibody library is one in which the patient(s) from which theantibodies were derived have been exposed to the antigen against whichthe domain antibody library will ultimately be screened. As an example,where the goal is to pan or screen the domain antibody library fordomain antibodies that neutralize an HIV antigen, an immune antibodylibrary would be one whereby the patient from which the antibodies weresampled was infected with HIV.

Alternatively, the source libraries can comprise a syntheticallyprepared CDR repertoire.

Conveniently, the mutagenesis and/or grafting of the CDRs may beachieved by the method of overlap extension using primers which containat each end sequences that are complementary or homologous to the anchorregions that form the basis of the framework region of m0 (or a variantthereof) and, in between, the CDR sequences. In one aspect, the primersof Table 1 can be used to obtain CDR products from any human antibodylibrary and/or introduce mutations in CDR1 (with the H1R primer, whichcontains four triplet regions of degenerate sequences (underlinedregions). Similarly mutations can be introduced in CDR2 and CDR3.

Regarding CDR mutagenesis, it is important when designing the CDRprimers also to take into account sequence homology within the CDRregions which was observed in the sequence data from the naive clones,as the amino acids concerned are thought to play a structural role inthe VH. It is desirable that highly conserved sequences within the CDRs,that is, residues that are conserved amongst a substantial proportion ofthe VH domains in the naive repertoire, should be retained in thesynthetically modified primers, and excluded as targets for mutagenesis.

Splicing by overlap extension is a modification of the polymerase chainreaction, which has been used to generate gene fusions at very specificpositions. It is based on the ability to fuse and amplify two DNAfragments containing homologous sequences i.e. ‘anchors’ around thefusion point.

For example, for the preparation of a ‘synthetic’ expression library,CDR primers incubated with framework region fragments will anneal attheir complementary ends and fuse to generate randomised framework-CDRencoding fragments. This process yields CDR-1/FR-2, CDR-2/FR-3 andCDR-3/FR-4 fusion fragments. Two of these fragments are then fused, andso forth.

There then follows a denaturation step after which the fragments can befurther annealed at the ‘anchor-regions’ and extended yielding thefused, double stranded gene product. If required this reaction can befollowed by the PCR reaction amplifying the quantity of fused genematerial. This method can easily be extended to fuse three or morefragments. Such or similar methods are disclosed in U.S. Pat. No.7,196,187, and U.S. published application No. 2007/0202105, each ofwhich are incorporated herein by reference.

Splicing by overlap extension allows the linking of the fusion fragmentsat specific positions to produce a fully assembled VH gene which can becloned into a suitable phage display vector, such as the vector pHEN.5,using restriction enzymes such as SfiI/NotI.

It will be appreciated that other methods of introducing mutations,preferably including random or partially random sequences, into the CDRswould also be applicable. Such methods include, for example, cassettemutagenesis or the use of error-prone ‘mutator’ strains as bacterialhosts.

Any suitable methods can then be used to evaluate the size and sequencediversity of the domain antibody library. It will be appreciated thatsize and sequence diversity are key features of high-quality libraries.A finding consistent with theoretical considerations is that theaffinity of antibodies selected is proportional to the size of thelibrary, with K_(d)s ranging from 10⁶⁻⁷ for the smaller libraries to 10⁹for the larger ones, and antibodies with affinity comparable to thoseobtained from immune libraries can be selected from naïve libraries thatare large enough (Andrew et al., J. Immunol. Methods, 2004). Forexample, random nucleotide sequencing can be performed on a randomselection of clones of the library to evaluate the source and extent ofsequence diversity of the CDRs of the library. Diverse antibodylibraries contain a high number of uniquely arranged VH sequences, i.e.a diverse assemblage of CDR1, CDR2 and CDR3 with unique combinations andsequences. Preferably, the domain antibody library of the inventioncomprises preferably at least about 10⁸, preferably at least about 10⁹,10¹⁰, more preferably about 10¹¹ or 10¹² unique whole VH sequences.

In addition, the expressed VH products of the library clones can beevaluated for physical properties, such as solubility, stability andtendency to aggregate. Such methods would require expression ofindividual clones in a suitable vector in a host cell, e.g. E. coli,followed by the isolation of the expressed product, followed then by ananalysis of the solubility, stability and tendency to aggregate, or anyother desire trait.

Once a domain antibody library has been constructed, the presentinvention contemplates the screening of the library for antibodies thatbind to or which are specifically immunoreactive against an antigen ofinterest, e.g. CD4i antigen.

The host cell used to express the VH products of the domain antibodylibrary (e.g. phagemid library) may be prokaryotic or eukaryotic but ispreferably bacterial, particularly E. coli.

The domain antibody library (e.g. phagemid library) according to theinvention may be screened for antigen binding activity usingconventional techniques well known in the art as described, for example,in Hoogenboom, Tibtech, 1997 (15), 62 70. By way of illustration,bacteriophage displaying a repertoire of nucleic acid sequencesaccording to the invention on the surface of the phage may be screenedagainst different antigens by a ‘panning’ process (see McCafferty, J.,Griffiths, A D, Winter, G. and Chiswell, D J, Nature, 348 (1990)552-554, which is incorporated herein by reference in its entirety)whereby the VH domains are screened for binding to an immobilizedantigen (e.g. antigen on magnetic bead or bottom of microtiter well).Non-binding phage are removed. Binding phage are retained, eluted andamplified in bacterial or other suitable host (depending on the vectorused). The panning cycle is repeated until enrichment of phage orantigen is observed and individual phage clones are then assayed forbinding to the panning antigen and to uncoated polystyrene by phageELISA.

As an indication of the binding affinities of antibodies that resultfrom the screening described in the invention, dissociation constantsfor the VHs recognizing a protein antigen will typically be less than100 nM, preferably less than 75 nM, more preferred less than 50 nM,still more preferred at less than 40 nM, most preferred less than 25 nM.

In one particular embodiment, a sequential panning technique is employedto obtain antibodies that bind to antigens from the same, butgenetically distinct viruses or organisms. This approach is advantageousin identifying antibodies that might be more cross-reactive betweendifferent genetically distinct isolates of the same virus or otherorganisms, e.g. different isolates of HIV-1 or HIV-1 viruses fromdifferent clades. Antibodies with broader cross reactivity againstdifferent isolates of the same target would be advantageous. It iscommonly known that in certain situations, e.g. HIV-1, it is difficultto obtain antibodies that will effective against HIV-1 isolates toogenetically distinct from the isolate against which the antibody wasprepared. That is, with some anti-HIV antibodies, the antibodies cansuffer from having too narrow of an effectiveness.

In the context of phage display technology, this can be done bysequentially changing the antigen during the panning of a phage displaylibrary of the invention. By sequentially changing the antigen duringpanning of phage display libraries and screening the panned librariesusing different antigens, the selected phage will display dAbs againstconserved epitopes shared among all antigens used during the entireselection process. In one embodiment, gp120 antigens or gp120/CD4complex antigens from two or more different HIV-1 isolates can besequentially changed during the panning of phage display libraries ofthe invention, which will result in the enrichment of phages thatdisplay dAbs having affinity to shared epitopes of both or each of thegp120 antigens used during panning Sequential panning of HIV antigens isdiscussed further in Zhang et al., 2004 and Zhang et al., 2003, each ofwhich are incorporated herein by reference. The herein Example 2 employsa sequential panning approach.

As an example, antibodies which bind to HIV CD4i can be identified usinga phagemid panning process. The HIV CD4i antigen may be coated on amicrotiter plate or a magnetic bead and incubated with the domainantibody phagemid library of interest. Phage-linked VHs that do not bindto the CD4i antigen may be washed from the plate, leaving only boundphage. The bound phage may be eluted by addition of a thiol reducingagent such as dithiotreitol (DTT) resulting in cleavage of the disulfidebond linking the antibody to the phage. The recovered population ofphage may be amplified by infection of E. coli hosts. This panningprocess may be repeated using the enriched population of phage tofurther enrich for a population of phage-linked antibodies that bind tothe HIV CD4i antigen. The gene sequence encoding the dAbs may then beexcised using standard cloning techniques and transferred to an E. coliexpression vector which is used to transform an E. coli expression cellline. dAbs from the enriched pool may then be expressed and purified andcharacterized.

Domain Antibodies

In another embodiment, the present invention provides novel domainantibodies. The domain antibodies of the invention are advantageous overprior art antibodies, particularly domain antibodies, because theinventive antibodies are more stable, more soluble and do not tend toform aggregates or polymerization products in solution. In addition, thedomain antibodies of the invention have high affinity for their targetepitopes, are highly expressed, possess strong antigen neutralizationaction, and optionally can be broadly cross-reactive, e.g.cross-reactive antibody against HIV isolates from different clades.Accordingly, in one aspect, the domain antibodies of the invention canbe used therapeutically to treat or prevent a number of conditions (e.g.cancer) or infections (e.g. HIV), or diagnostically to diagnose ordetect condition- or infection-related antigens. As noted above, theadvantageous features of the inventive antibodies of the invention stemat least in part to the novel VH framework, m0, and its derivatives, ofthe invention that forms the basis of the dAb library from which theinventive antibodies can be obtained.

In one aspect, the present invention embodies a number of differentantibodies having gp120/CD4 CD4i binding characteristics identified byscreening the m0 based domain antibody library of the invention using asequential panning approach. In one aspect, the present inventionprovides a novel dAb, designated as m36, which possesses goodsolubility, has no detectable oligomerization tendencies, is highlyspecific for the CD4i antigen, and has broad affinity for different HIVisolates from different clades. (See Example 2). The amino acid andnucleotide sequence of m36 are given in FIGS. 18 and 19. The CDRsequences and framework sequences of m36 are shown in FIG. 18.

The present invention also contemplates, in accordance with the methodspreviously described or any other suitable known methods, derivatives ofm36, which have been derivatived in any advantageous manner, such as,introducing changes in the at the nucleotide sequence level of theframework and/or the CDR1, CDR2 or CDR3 of m36. Methods for introducinggenetic change to the nucleotide sequence of m36 are well known in theart, and can include PCR-based random or site-directed mutagenesis,insertions, deletions, gene shuffling, CDR grafting, etc. The presentinvention particular contemplates dAbs that have framework and/or CDRamino acid sequences having at least 60%, preferably at least 80%, or85%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% or greatersequence identity with the framework and/or CDR sequences of m36.

Domain antibodies and/or fragments or derivatives thereof of theinvention may be purified from any cell that expresses the antibodies,including host cells that have been transfected with antibody-encodingexpression constructs. The host cells may be cultured under conditionswhereby the antibodies are expressed. Purified dAbs antibodies may beseparated from other cellular components that may associate with theantibodies in the cell, such as certain proteins, carbohydrates, orlipids using methods well known in the art. Such methods include, butare not limited to, size exclusion chromatography, ammonium sulfatefractionation, ion exchange chromatography, affinity chromatography, andpreparative gel electrophoresis. Purity of the preparations may beassessed by any means known in the art, such as SDS-polyacrylamide gelelectrophoresis.

Alternatively, antibodies may be produced using chemical methods tosynthesize its amino acid sequence or portions of the antibody sequence(e.g. CDR sequences), such as by direct peptide synthesis usingsolid-phase techniques (e.g., Merrifield, J. Am. Chem. Soc.85:2149-2154, 1963; Roberge, et al., Science 269:202-204, 1995, each ofwhich are incorporated herein by reference). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Optionally, fragments of antibodies may beseparately synthesized and combined using chemical methods to produce afull-length molecule.

In embodiments where the VH framework is obtained from a non-humananimal and used to construct a dAb library, methods for humanization arecontemplated. It will be generally appreciated by those skilled in theart that the most critical determinants of antibody selectivity andbinding affinity are the sequences and resulting conformations of thecomplementarity regions (CDRs). Whole antibodies (e.g. human IgG)contain six CDRs, three within the heavy chain variable region (VH) andthree within the light chain variable region (VL), for each Fab arm ofthe complete molecule. The intervening sequences between the CDRs withinVH and VL are the framework regions which spatially orient the CDRs. TheCDRs together form the antigenic binding sites within antibodies. Thecritical role of these CDRs in determining the functional properties ofantibodies has long been exploited in the processes of antibodyhumanization and antibody optimization. In the former process, the CDRsfrom a monoclonal antibody, for example, a mouse antibody, aretransferred to a human antibody of similar framework design therebyresulting in an antibody with the same functional properties and reducedimmunogenicity in man.

The success of this process is evident from the number of humanizedantibodies that have been successfully commercialized as humantherapeutics and include HERCEPTIN® (trastuzumab, Genentech, Inc., SouthSan Francisco, Calif.), SYNAGIS® (palivizumab, Medimmune, Inc.,Gaithersburg, Md.), CAMPATH® (alemtuzumab, Genzyme Oncology, Cambridge,Mass.), ZENAPAX® (daclizumab, Roche Pharmaceuticals, Nutley, N.J.),XOLAIR® (omalizumab, Genentech, Inc., South San Francisco, Calif.),RAPTIVA® (efalizumab, Genentech, Inc., South San Francisco, Calif.),AVASTIN® (bevacizumab, Genentech, Inc., South San Francisco, Calif.),and MYLOTARG® (gemtuzumab ozogamicin, Wyeth-Ayerst, Madison, N.J.).Other examples have been described in Singer, et al., (J. Immunol,150:2844-2857, 1993); Luo, et al., (J. Immunol Meth., 275:31-40, 2002);and Kostelny, et al., (Int. J. Cancer 93; 556-565, 2001).

In principal, a framework sequence from any human antibody may serve asthe template for CDR grafting. However, it has been demonstrated thatstraight CDR replacement onto such a framework often leads tosignificant loss of binding affinity to the antigen (Glaser, et al., J.Immunol. 149:2606, 1992); Tempest, et al., Biotechnology 9:266, 1992;Shalaby, et al., J. Exp. Med. 17: 217, 1992). The more homologous ahuman antibody is to the original antibody, the less likely combiningthe CDRs with the human framework will be to introducing distortionsinto the CDRs that could reduce affinity. In view of this generalprinciple, it is quite unexpected then that the VH framework of the dAblibrary of the invention is compatible in terms of domain folding andsolubility with such a highly diverse repertoire of CDRs. (See Example2).

Domain Antibody Modifications

In yet another embodiment, the present invention relates to modifieddAbs, and provides methods for modifying domain antibodies identified inaccordance with the invention. The modifications may be geneticmodifications to the nucleic acid encoding a domain antibody polypeptideof the invention or they may be chemical, structural, or physicalmodifications made directly to an isolated domain antibody of theinvention to impart additional advantageous properties to a domainantibody of the invention regarding, for example, the level ofexpression, stability, solubility, epitope affinity, antigenneutralization activity, or penetration characteristics, etc.

In one aspect, the present invention contemplates introducing geneticmodifications into one or more CDRs or to the framework sequence of thedomain antibodies of the invention. Such genetic modifications canconfer additional advantageous characteristics, i.e. geneticoptimization, of the domain antibodies identified from libraryscreening, including, for example, enhanced solubility, enhancedaffinity, and enhanced stability. Any type of genetic modification iscontemplated by the present invention, including, for example,site-directed mutagenesis, random mutagenesis, insertions, deletions,CDR grafting (i.e. genetic replacement of one CDR for another CDR), andthe construction and/or preparation of fusion proteins between domainantibodies of interest and desired fusion partners, e.g., serumalbumin-binding peptide (SaAb), which was unexpectedly found to increasethe stability of the dAbs of the invention, or soluble CD4, which wasunexpectedly found to synergistically impact the neutralization capacityof some of the domain antibodies of the invention. All of thesetechniques are well known to those skilled in the art. See Ausubel etal., Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, 2000, incorporated herein by reference. Reference to CDR graftingcan be made to Nicaise, et al., Protein Science 13:1882-1891, 2004. Theeffect of any genetic modification can be tested or screened withoutundue experimentation using any of the methods described herein or othermethods already known to one of ordinary skill in the art. For example,affinity of a domain antibody to a target antigen can be assessed usingthe herein described BIA procedure.

In another aspect, other modifications contemplated by the presentinvention relate to chemical modifications of the domain antibodies ofthe invention to confer additional advantageous features, such asenhanced stability and/or solubility and/or half-life.

In one particular aspect, the domain antibodies of the present inventioncan be PEGylated, or coupled to polymers of similar structure, functionand purpose (“PEG or PEG-like polymers”), to confer enhanced stabilityand half-life. PEGylation can provide increased half-life and resistanceto degradation without a loss in activity (e.g. binding affinity)relative to non-PEGylated antibody polypeptides. The skilled artisanwill appreciate, however, that PEGylation may not be advantageous withrespect to some targets, in particular, those epitopes which aresterically-obstructed, e.g. CD4i epitope. Thus, in cases where theinventive domain antibody targets a size-restricted epitope, the domainantibody should minimally PEGylated so as not to negatively impact theaccessibility of the antibody to the size-restricted antigen. Theskilled artisan will appreciate that this general principle should beapplied to any modifications made to the dAbs of the invention.

Any method known in the art to couple the domain antibodies of theinvention to PEG or PEG-like polymers is contemplated by the presentinvention. PEG or PEG-like moieties which can be utilized in theinvention can be synthetic or naturally occurring and include, but arenot limited to, straight or branched chain polyalkylene, polyalkenyleneor polyoxyalkylene polymers, or a branched or unbranched polysaccharide,such as a homo- or heteropolysaccharide. Preferred examples of syntheticpolymers which can be used in the invention include straight or branchedchain poly(ethylene glycol) (PEG), polypropylene glycol), or poly(vinylalcohol) and derivatives or substituted forms thereof. Substitutedpolymers for linkage to the domain antibodies of the invention can alsoparticularly include substituted PEG, including methoxy(polyethyleneglycol). Naturally occurring polymer moieties which can be used inaddition to or in place of PEG include, for example, lactose, amylose,dextran, or glycogen, as well as derivatives thereof which would berecognized by persons skilled in the art.

PEGylation of the domain antibodies of the invention may be accomplishedby any number of means (see for example Kozlowski-A & Harris-J M (2001)Journal of Controlled Release 72:217). PEG may be attached to the domainantibody construct either directly or by an intervening linker.Linkerless systems for attaching polyethylene glycol to proteins isdescribed in Delgado et al., (1992), Crit. Rev. Thera. Drug Carrier Sys.9:249-304 Francis et al., (1998), Intern. J. Hematol. 68:1-18; U.S. Pat.No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO 98/32466,the disclosures each of which are incorporated herein by reference. Thefirst step in the attachment of PEG or other polymer moieties to thedomain antibody construct of the invention typically is the substitutionof the hydroxyl end-groups of the PEG polymer by electrophile-containingfunctional groups. Particularly, PEG polymers are attached to eithercysteine or lysine residues present in the domain antibody constructmonomers or multimers. The cysteine and lysine residues can be naturallyoccurring, or can be engineered into the domain antibody molecule.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monomethoxy polyethyleneglycol (MPEG) using tresylchloride. Following reaction of amino acidresidues with tresylated MPEG, polyethylene glycol is directly attachedto the amine groups. Thus, the invention includesprotein-polyethyleneglycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoroethane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber of additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference.

Other derivatized forms of polymer molecules include, for example,derivatives which have additional moieties or reactive groups presenttherein to permit interaction with amino acid residues of the domainantibodies described herein. Such derivatives includeN-hydroxylsuccinimide (NHS) active esters, succinimidyl propionatepolymers, and sulfhydryl-selective reactive agents such as maleimide,vinyl sulfone, and thiol. The reactive group (e.g., MAL, NHS, SPA, VS,or Thiol) may be attached directly to the PEG polymer or may be attachedto PEG via a linker molecule.

The size of polymers useful in the invention can be in the range of 500Da to 60 kDa, for example, between 1000 Da and 60 kDa, 10 kDa and 60kDa, 20 kDa and 60 kDa, 30 kDa and 60 kDa, 40 kDa and 60 kDa, and up tobetween 50 kDa and 60 kDa. The polymers used in the invention,particularly PEG, can be straight chain polymers or may possess abranched conformation.

The present invention also contemplates the coupling (either by physicalattachment of separate molecules, or through the use of geneticengineering to construct a fusion protein between an antibody of theinvention (e.g., m36) and an adduct protein (e.g., serum albumin-bindingpeptide) of adduct molecules, which can be various polypeptides orfragments thereof which occur naturally in vivo and which resistdegradation or removal by endogenous mechanisms. Molecules whichincrease half life may be selected from the following: (a) proteins fromthe extracellular matrix, eg. collagen, laminin, integrin andfibronectin; (b) proteins found in blood, e.g., serum albumin, serumalbumin-binding peptide (SAbp—e.g., see the fusion prepared inaccordance with Example 3), fibrinogen A, fibrinogen B, serum amyloidprotein A, heptaglobin, protein, ubiquitin, uteroglobulin, β-2microglobulin, plasminogen, lysozyme, cystatin C, alpha-1-antitrypsinand pancreatic kypsin inhibitor; (c) immune serum proteins, e.g. IgE,IgG, IgM and their fragments e.g. Fc; (d) transport proteins, e.g.retinol binding protein; (e) defensins, e.g. beta-defensin 1, neutrophildefensins 1, 2 and 3; (f) proteins found at the blood brain barrier orin neural tissues, e.g. melanocortin receptor, myelin, ascorbatetransporter; (g) transferrin receptor specificligand-neuropharmaceutical agent fusion proteins, brain capillaryendothelial cell receptor, transferrin, transferrin receptor, insulin,insulin-like growth factor 1 (IGF 1) receptor, insulin-like growthfactor 2 (IGF 2) receptor, insulin receptor; (h) proteins localised tothe kidney, e.g. polycystin, type IV collagen, organic anion transporterK1, Heymann's antigen; (i) proteins localized to the liver, e.g. alcoholdehydrogenase, G250; (j) blood coagulation factor X; (k) α-1antitrypsin; (1) HNF 1 α.; (m) proteins localised to the lung, e.g.secretory component (binds IgA); (n) proteins localised to the heart,eg. HSP 27; (o) proteins localised to the skin, eg, keratin; (p) bonespecific proteins, such as bone morphogenic proteins (BMPs) e.g. BMP-2,-4, -5, -6, -7 (also referred to as osteogenic protein (OP-1) and -8(OP-2); (q) tumour specific proteins, eg. human trophoblast antigen,herceptin receptor, oestrogen receptor, cathepsins eg cathepsin B (foundin liver and spleen); (r) disease-specific proteins, eg. antigensexpressed only on activated T-cells: including LAG-3 (lymphocyteactivation gene); osteoprotegerin ligand (OPGL) see Kong Y Y et alNature (1999) 402, 304-309; OX40 (a member of the TNF receptor family,expressed on activated T cells and the only costimulatory T cellmolecule known to be specifically up-regulated in human T cell leukaemiavirus type-I (HTLV-I)-producing cells—see Pankow R et al J. Immunol.(2000) Jul. 1; 165(1):263-70; metalloproteases (associated witharthritis/cancers), including CG6512 Drosophila, human paraplegin, humanFtsH, human AFG3L2, murine ftsH; angiogenic growth factors, includingacidic fibroblast growth factor (FGF-1), basic fibroblast growth factor(FGF-2), Vascular endothelial growth factor/vascular permeability factor(VEGF/VPF), transforming growth factor-α (TGF-α), tumor necrosisfactor-alpha (TNF-α), angiogenin, interleukin-3 (IL-3), interleukin-8(IL-8), platelet derived endothelial growth factor (PD-ECGF), placentalgrowth factor (PlGF), midkine platelet-derived growth factor-BB (PDGF),fractalkine; (s) stress proteins (heat shock proteins); and (t) proteinsinvolved in Fc transport.

In a particular aspect, an antibody of the invention, e.g., m36, can becoupled with CD4 or a fragment or mimic thereof to increase theeffectiveness of the binding of the antibody with its cognate HIVepitope, e.g., a CD4i epitope. It was also been surprisingly found thatsoluble CD4 or fragments or mimics thereof can be co-administered withan antibody of interest of the invention (e.g. m36) to synergisticallyimprove the effectiveness of the antibody's neutralization capability.(see Example 3 for further discuss). CD4 mimics are known in the art andcan be found described, for example, in U.S. Publication Nos.2006/0073576, 2008/0096187, each of which are incorporated herein byreference.

In another aspect, the domain antibodies of the invention may bemultimerized, as for example, hetero- or homodimers, hetero- orhomotrimers, hetero- or homotetramers, or higher order hetero- orhomomultimers. Multimerisation can increase the strength of antigenbinding, wherein the strength of binding is related to the sum of thebinding affinities of the multiple binding sites. The domain antibodiescan be multimerized in another aspect by binding to an additional one,two, three or more polypeptide which function to stabilize the dAbagainst degradation. Such polypeptides may include common bloodproteins, such as, albumin, or fragments thereof. Example 3 discussesthe construction of m36CH3 as an example of preparing a multimerizedantibody of the invention.

In certain aspects, linker may be used to join (either through physicalcoupling or through a genetic engineering approach) an antibody of theinvention, e.g., m36, with a suitable or appropriate adduct molecule orother desired fusion partner. As defined herein, a “fusion partner” canbe any molecule, such as an adduct molecule for enhancing the stabilityof an antibody, that is fused through recombinant means or throughphysical means to an antibody of interest of the invention.

In yet another aspect, modifications relating to enhancing or modifyingantibody activity are contemplated by the present invention. Forexample, it may be desirable to modify the antibody of the inventionwith respect to effector function, so as to enhance the effectiveness ofthe antibody in treating a condition, infection or disorder. For examplecysteine residue(s) may be introduced in the domain antibodypolypeptide, thereby allowing interchain disulfide bond formation in amultimerized form of the inventive antibodies. The homodimeric orheterodimeric (or multimeric) antibodies may include combinations of thesame domain antibody polypeptide chains or different domain antibodypolypeptide chains, such that more than one epitope is targeted at atime by the same construct. Such epitopes can be proximally located inthe target (e.g. on the HIV target) such that the binding of one epitopefacilitates the binding of the multmeric antibody of the invention tothe second or more epitopes. The epitopes targeted by multimericantibodies can also be distally situated.

The invention also contemplates modifying the domain antibodies of theinvention to form immunoconjugates comprising the domain antibodies ofthe invention conjugated to cytotoxic agents, such as a chemotherapeuticagents, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant or animal origin, or fragments thereof), radioactive isotopes(i.e., a radioconjugate), or antiviral compounds (e.g. anti-HIVcompounds).

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term can include radioactive isotopes (e.g., I₁₃₁, I₁₂₅, Y₉₀and Re₁₈₆), chemotherapeutic agents, and toxins such as enzymaticallyactive toxins of bacterial, fungal, plant or animal origin, or fragmentsthereof.

A “chemotherapeutic agent” is a type of cytotoxic agent useful in thetreatment of cancer. Examples of chemotherapeutic agents includeAdriamycin, Doxorubicin, 5-Fluorouracil, Cytosine arabinoside (“Ara-C”),Cyclophosphamide, Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin,Taxol, Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,Aminopterin, Dactinomycin, Mitomycins, Esperamicins, Melphalan and otherrelated nitrogen mustards.

The invention also contemplates immunoconjugation with enzymaticallyactive toxins or fragments thereof. Enzymatically active toxins andfragments thereof which can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes.

Where the inventive antibodies are intended to target viruses, bacteriaor other pathogens, the invention also contemplates immunoconjugation ofthe domain antibodies with anti-viral, anti-bacterial or other chemicalsand/or compounds that might improve or increase the effectiveness of thedomain antibodies of the invention against intended targets, such as,for example, HIV.

For example, the inventive antibodies can be immunoconjugated, or in thealternative, co-administered with, an antibacterial compound, such as,for example, a macrolide (e.g., tobramycin (TOBI®)), a cephalosporin(e.g., cephalexin (KEFLEX®), cephradine (VELOSEF®), cefuroxime(CEFTIN®), cefprozil (CEFZIL®), cefaclor (CECLOR®), cefixime (SUPRAX®)or cefadroxil (DURICEF®), a clarithromycin (e.g., clarithromycin(BIAXIN®)), an erythromycin (e.g., erythromycin (EMYCIN®)), a penicillin(e.g., penicillin V (V-CILLIN K® or PEN VEE K®)) or a quinolone (e.g.,ofloxacin (FLOXIN®), ciprofloxacin (CIPRO®) or norfloxacin (NOROXIN®)),aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins,butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin,paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicolantibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, andthiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin),carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem andimipenem), cephalosporins (e.g., cefaclor, cefadroxil, cefamandole,cefatrizine, cefazedone, cefozopran, cefpimizole, cefpiramide, andcefpirome), cephamycins (e.g., cefbuperazone, cefmetazole, andcefminox), monobactams (e.g., aztreonam, carumonam, and tigemonam),oxacephems (e.g., flomoxef, and moxalactam), penicillins (e.g.,amdinocillin, amdinocillin pivoxil, amoxicillin, bacampicillin,benzylpenicillinic acid, benzylpenicillin sodium, epicillin,fenbenicillin, floxacillin, penamccillin, penethamate hydriodide,penicillin o-benethamine, penicillin 0, penicillin V, penicillin Vbenzathine, penicillin V hydrabamine, penimepicycline, andphencihicillin potassium), lincosamides (e.g., clindamycin, andlincomycin), amphomycin, bacitracin, capreomycin, colistin, enduracidin,enviomycin, tetracyclines (e.g., apicycline, chlortetracycline,clomocycline, and demeclocycline), 2,4-diaminopyrimidines (e.g.,brodimoprim), nitrofurans (e.g., furaltadone, and furazolium chloride),quinolones and analogs thereof (e.g., cinoxacin, clinafloxacin,flumequine, and grepagloxacin), sulfonamides (e.g., acetylsulfamethoxypyrazine, benzylsulfamide, noprylsulfamide,phthalylsulfacetamide, sulfachrysoidine, and sulfacytine), sulfones(e.g., diathymosulfone, glucosulfone sodium, and solasulfone),cycloserine, mupirocin and tuberin.

In another example, the inventive antibodies can be immunoconjugated, orin the alternative, co-administered with, an antiviral compound, suchas, for example, a zidovudine, acyclovir, gangcyclovir, vidarabine,idoxuridine, trifluridine, and ribavirin, as well as foscarnet,amantadine, rimantadine, saquinavir, indinavir, amprenavir, lopinavir,ritonavir, adefovir, clevadine, entecavir, and pleconaril.

Methods for modifying the domain antibodies of the invention with thevarious cytoxic agents, chemotherapeutic agents, toxins, antibacterialcompounds, and antiviral compounds, etc. mentioned above are well knownin the art. For example, immunoconjugates of the antibody and cytotoxicagents can be made using a variety of bifunctional protein couplingagents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCL), active esters (such as disuccinimidylsuberate), aldehydes (such as glutareldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

The domain antibodies can also be modified with useful detectableagents, such as, for example, fluorescent compounds. Exemplaryfluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. The domain antibody construct mayalso be derivatized with detectable enzymes such as alkalinephosphatase, horseradish peroxidase, glucose oxidase and the like. Whenthe domain antibody construct is derivatized with a detectable enzyme,it is detected by adding additional reagents that the enzyme uses toproduce a detectable reaction product. The domain antibody construct mayalso be derivatized with biotin, and detected through indirectmeasurement of avidin or streptavidin binding.

The skilled artisan will appreciate it may be advantageous to couple anyof the aforementioned molecular entities to the domain antibodies of theinvention through flexible linkers, such as flexible polypeptide chains.Such linkers may be required to avoid a loss in activity of the domainantibodies, or to avoid sterically restricting the domain antibodiessuch that they lose their effectiveness in binding to cognate epitopes,in particular, those epitopes which themselves may be stericallyrestricted, e.g. HIV CD4i epitopes.

Another type of covalent modification contemplated by the presentinvention involves chemically or enzymatically coupling glycosides tothe domain antibodies of the invention. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston, CRC Crt. Rev. Biochem., pp. 259-306 (1981).

Removal of any carbohydrate moieties present on the antibodies of theinvention may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal. Arch. Biochem. Biophys. 259:52 (1987) and by Edge et al. Anal.Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties onantibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al. Meth. Enzymol. 138:350(1987).

Antigens

The present invention, in another embodiment, relates to antigens ofinterest that may be used to screen the domain antibody library of theinvention to identify useful and effective domain antibodies of theinvention.

In one preferred aspect, the antigens of the invention are HIV antigens.The antigens can be from any HIV isolate, e.g. 92UG, Bal, JR-FL, JRCSF,IIIB, 89.6, R2, NL4-3, GXG, Z2Z6, or GXE. The HIV source of antigens canalso be from any known clade of HIV, including clade A, B, C, D or E. Itwill be appreciated that HIV is different in structure from otherretroviruses. It is about 120 nm in diameter and roughly spherical. Itis composed of two copies of positive single-stranded RNA that codes fornine genes enclosed by a conical capsid composed of 2,000 copies of theviral protein p24. The single-stranded RNA is tightly bound tonucleocapsid proteins, p7 and enzymes needed for the development of thevirion such as reverse transcriptase, proteases, ribonuclease andintegrase. A matrix composed of the viral protein p17 surrounds thecapsid ensuring the integrity of the virion particle. The viralenvelope, which forms the outer shell of the virus, is composed of aphospholipid bilayer derived from the membrane of a human cell whenoutwardly budding from the cell. Embedded in the viral envelope areproteins from the host cell and about 70 copies of the Env “spike”complex. Env consists of a cap made of three molecules calledglycoprotein (gp) 120 (gp120) and a stem consisting of three gp41molecules that anchor the structure into the viral envelope. Thisglycoprotein complex enables the virus to attach to and fuse with targetcells to initiate the infectious cycle.

Regarding the HIV genome, of the nine genes that are encoded within theRNA genome, three of these genes, gag, pol, and env, contain informationneeded to make the structural proteins for new virus particles. Forexample, env codes for a protein called gp160 that is broken down by aviral enzyme to form gp120 and gp41. The six remaining genes, tat, rev,nef, vif, vpr, and vpu (or vpx in the case of HIV-2), are regulatorygenes for proteins that control the ability of HIV to infect cells,replicate, or cause disease. The protein encoded by nef, for instance,appears necessary for the virus to replicate efficiently, and thevpu-encoded protein influences the release of new virus particles frominfected cells. The ends of each strand of HIV RNA contain an RNAsequence called the long terminal repeat (LTR). Regions in the LTR actas switches to control production of new viruses and can be triggered byproteins from either HIV or the host cell.

The HIV antigens can be any of those indicated above, and especially,those for which the neutralization of by antibody would inhibit or ceasethe virus's ability to replicate, ability to infect cells, ability toassemble, or its ability to process its viral genetic or proteinmaterial, etc. In one embodiment, the antigen is the Env proteincomplex. It will be appreciated that the primary neutralization targeton the HIV-1 virion is the envelope glycoprotein (Env), which promotesvirus entry by catalyzing fusion between the virion and target cellmembranes. Env is a major focus for humoral vaccine and antibody-basedimmunotherapeutic strategies against HIV-1 (see Parren et al., 2001;Wyatt et al., 1998, for further detail regarding strategies forneutralization by anti-Env antibodies, each of which are incorporatedherein by reference). One problem in the art, however, is that effortsto develop effective neutralizing antibodies against Env have beenfrustrated by the difficulties in eliciting antibodies with potentneutralizing activities against genetically diverse HIV-1 isolates. (Deyet al., 2003).

In a preferred embodiment, the antigen is an inducible antigen. As usedherein, the term “inducible antigen” or “inducible epitope” refers tothose antigens or epitopes which are present on a polypeptide which,initially are sterically unavailable for antibody interaction, butthrough the binding of the polypeptide to a second interacting molecule,such as a drug, second polypeptide, ligand, receptor or nucleic acid,becomes exposed as a result of an induced conformational change in thepolypeptide caused by the interaction between the polypeptide and thesecond interacting molecule. In one particular aspect, the inducibleantigen is CD4i, which is an antigen comprising an epitope on gp120, theexposure of which is induced by binding to its cognate cellularreceptor, CD4. See Labrijn et al., 2003, which is incorporated herein byreference.

While not wishing to be bound by theory, the skilled artisan willappreciate that HIV-1 entry into host cells is initiated by the bindingof the gp120 subunit of the viral Env complex (the “spike” complex) tothe host cell receptor (CD4). This interaction induces conformationalchanges in gp120 resulting in the exposure of a conserved high-affinitybinding site for the coreceptor (the chemokine receptors CCR5 or CXCR4)(Sattentau et al., 1991; Sattentau et al., 1993).

A second binding step between the gp120-CD4 complex and the coreceptoris then thought to induce additional conformational changes thatultimately result in the fusion of viral and host cell membranes(Dimitrov, 2004; Jones et al., 1991). Not wishing to be bound by theory,neutralizing antibodies are believed to act, at least in part, bybinding to the exposed Env surface and obstructing the initialinteraction between a trimeric array of gp120 molecules on the virionsurface and receptor molecules on the target cell.

In response, HIV-1 has evolved a number of strategies to evaderecognition by neutralizing antibodies, particularly those directed tothe conserved CD4 and coreceptor binding sites of Env. The extent ofprotection of these sites from antibody recognition is limited by thenecessity to preserve the accessibility for receptor interaction. In thecase of the binding site of CD4 to gp120 (the “CD4bs”), this has led tothe following structural features: (i) it is partially obscured fromantibody recognition by the V1/V2 loop and associated carbohydratestructures; (ii) the flanking residues are variable and modified byglycosylation; (iii) it is recessed to an extent that limits directaccess by an antibody variable region; (iv) clusters of residues withinthe CD4bs that do not directly interact with CD4 are subject tovariation among virus strains; (v) many gp120 residues interact with CD4via main-chain atoms, allowing for variability in the correspondingamino acid side chains; and (vi) there is considerable conformationalflexibility within the CD4-unbound state of gp120, and antibody bindingtherefore requires relatively large entropic decreases, thus“conformationally masking” the conserved CD4bs (Kwong et al., 2002;Myszka et al., 2000).

The coreceptor binding site on gp120 is thought to be composed of ahighly conserved element on the β19 strand and parts of the V3 loop(Labrijn et al., 2003). These elements are masked by the V1/V2 variableloops in the CD4-unbound state and largely unavailable for antibodybinding (Labrijn et al., 2003). Upon CD4 binding, conformational changesare induced. These changes include displacement of the V1/V2 stem-loopstructure and consequent exposure of the coreceptor binding site(Labrijn et al., 2003). Binding studies with variable loop-deletedmutants suggest that CD4 induces additional rearrangement orstabilization of the gp120 bridging sheet near the 19 strand to form thefinal coreceptor binding surface (Labrijn et al., 2003). Since thebinding to CD4 occurs at the virus-cell interface, the exposedcoreceptor binding site is optimally positioned for interaction with thecoreceptor.

A highly conserved discontinuous structure on gp120 associated with thecoreceptor binding site is recognized by monoclonal antibodies (MAbs)that bind better to gp120 upon ligation with CD4. These so-calledCD4-induced (CD4i) antibodies, such as 17b and 48d (Labrijn et al.,2003), recognize a cluster of gp120 epitopes that are centered on theB19 strand and partially overlap the coreceptor binding site (Labrijn etal., 2003). Although such CD4i MAbs can neutralize some T-cell lineadapted HIV-1 strains, they are generally poorly neutralizing forprimary isolates (Labrijn et al., 2003; Poignard et al., 2001).Recently, an antibody Fab fragment, X5, from a phage display library anddirected to a CD4i epitope, neutralized a wide variety of primaryisolates (Labrijn et al., 2003; Moulard et al., 2002). Also recently,the Fab X5 antibody fragment was studied against other CD4i Mabs at amolecular level and found the smaller fragments were more able toneutralize different HIV-1 isolates (Labrijn et al., 2003).

Accordingly, in one embodiment, the domain antibodies of the presentinvention bind to or are specifically immunoreactive against the HIV-1CD4i epitope.

Analytical and Preparative Methods

Once an antibody in accordance with the invention is identified orobtained, for example, by any of the methods herein described, forexample, including by panning of the dAb library of the invention andexpressing same in a host, it may be preferable to carry out furthersteps to characterize and/or purify and/or modify the antibody. Forexample, it may be desirable to prepare a purified, high-titercomposition of the desirable antibody or to test the immunoreactivity ofthe identified antibody. The present invention contemplates any knownand suitable methods for characterizing, purifying, or assaying theantibodies of the present invention and it is expected the any person ofordinary skill in the art to which the invention pertains will have therequisite level of technical know-how and resources, e.g. technicalmanuals or treatises, to accomplish any further characterization,purification and/or assaying of the antibodies of the invention withoutundue experimentation.

For example, any useful means to describe the strength of binding (oraffinity) between a domain antibody of the invention and an antigen ofthe invention (e.g. CD4i antigen) can be used. For example, thedissociation constant, K_(d)(K_(d)=k2/k1=[antibody][antigen]/[antibody-antigen complex]) can bedetermined by standard kinetic analyses that are known in the art. Itwill be appreciated by those of ordinary skill in the art that thedissociation constant indicates the strength of binding between anantibody and an antigen in terms of how easy it is to separate thecomplex. If a high concentration of antibody and antigen are required toform the complex, the strength or affinity of binding is low, resultingin a higher K_(d). It follows that the smaller the K_(d) (as expressedin concentration units, e.g. molar or nanomolar), the stronger thebinding.

Affinity can be assessed and/or measured by a variety of knowntechniques and immunoassays, including, for example, enzyme-linkedimmunospecific assay (ELISA), Bimolecular Interaction Analysis (BIA)(e.g., Sjolander and Urbaniczky, Anal. Chem. 63:2338-2345, 1991; Szabo,et al., Curr. Opin. Struct. Biol. 5:699-705, 1995, each incorporatedherein by reference), and fluorescence-activated cell sorting (FACS) forquantification of antibody binding to cells that express antigen. BIA isa technology for analyzing biospecific interactions in real time,without labeling any of the interactants (e.g., BIACORE™). BIAcore isbased on determining changes in the optical phenomenon surface plasmonresonance (SPR) in real-time reactions between biological molecules,such as, an antibody of the invention and an antigen of interest, e.g.CD4i. References relating to BIAcore technology can be further found inU.S. Published Application Nos: 2006/0223113, 2006/0134800,2006/0094060, 2006/0072115, 2006/0019313, 2006/0014232, and2005/0199076, each of which are incorporated herein in their entiretiesby reference.

The domain antibodies of the invention may be assayed for immunospecificbinding by any suitable method known in the art. Assays involving anantibody and an antigen are known as “immunoassays,” which can beemployed in the present invention to characterize both the antibodiesand the antigens of the invention. The immunoassays which can be usedinclude but are not limited to competitive and non-competitive assaysystems using techniques such as western blots, radioimmunoassays, ELISA(enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al., eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety) andcan be performed without undue experimentation.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer; blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ₃₂P or ₁₂₅I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1, which is incorporated herein byreference.

ELISAs typically comprise preparing antigen, coating the well of a 96well microtiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1, which is incorporatedherein by reference.

Any suitable method for purifying antibodies is contemplated herein. Forexample, chromatographic methods, such as, for example, immuno-affinitychromatography (immobilized ligand to bind and trap antibody ofinterest), affinity chromatography, protein precipitation, ion exchangechromatography, hydrophobic interaction chromatography, size-exclusionchromatography, as well as electrophoresis, can be found described inthe technical literature, for example, in Methods in Enzymology, Volume182, Guide to Protein Purification, Eds. J. Abelson, M. Simon, AcademicPress, 1^(st) Edition, 1990, which is incorporated herein by reference.Thus, suitable materials for performing such purification steps, such aschromatographic steps, are known to those skilled in the art. Suchmethods are suitable for purification of any of the antibodies, antigensor any fragments thereof that are in accordance with the invention asdescribed herein.

Certain embodiments may require the purification or isolation ofexpressed proteins or antibodies or fragments thereof from a host cellor a portion thereof. Conventional procedures for isolating recombinantproteins from transformed host cells are contemplated by the presentinvention. Such methods include, for example, isolation of the proteinor fragments of interest by initial extraction from cell pellets or fromcell culture medium, followed by salting-out, and one or morechromatography steps, including aqueous ion exchange chromatography,size exclusion chromatography steps, high performance liquidchromatography (HPLC), and affinity chromatography may be used toisolate the recombinant protein or fragment. Guidance in the proceduresfor protein purification can be found in the technical literature,including, for example, Methods in Enzymology, Volume 182, Guide toProtein Purification, Eds. J. Abelson, M. Simon, Academic Press, 1^(st)Edition, 1990, which is already incorporated by reference.

Nucleic Acid Molecules, Vectors, and Host Cells

In another aspect, the present invention relates to nucleic acidmolecules, e.g. the phagemid clones of the invention, comprising thenucleotide sequences encoding the dAbs of the invention. These nucleicacid molecules may be used, for example, to express quantities of theantibodies for therapeutic or diagnostic use.

Nucleic acid molecules, e.g. phagemid clones, of the present inventionmay be isolated from host cells, free of other cellular components suchas membrane components, proteins, and lipids according to any known orsuitable method in the art. Nucleic acid molecules may be isolated usingstandard nucleic acid purification techniques, or synthesized using anamplification technique such as the polymerase chain reaction (PCR), orby using an automatic synthesizer. Methods for isolating polynucleotidesare routine and are known in the art. Any such technique for obtaining apolynucleotide may be used to obtain isolated polynucleotides encodingantibodies of the invention. For example, restriction enzymes and probesmay be used to isolate polynucleotides which encode antibodies.

Antibody-encoding cDNA molecules may be made with standard molecularbiology techniques, using mRNA as a template. Thereafter, cDNA moleculesmay be replicated using molecular biology techniques known in the artand disclosed in manuals such as Sambrook, et al., (Molecular Cloning: ALaboratory Manual, (Second Edition, Cold Spring Harbor Laboratory Press;Cold Spring Harbor, N.Y.; 1989, Vol. 1-3, incorporated herein byreference). An amplification technique, such as PCR, may be used toobtain additional copies of the polynucleotides.

To express a polynucleotide encoding an antibody, the polynucleotide maybe inserted into an expression vector that contains the necessaryelements for the transcription and translation of the inserted codingsequence. Methods that are well known to those skilled in the art may beused to construct expression vectors containing sequences encodingantibodies and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described, for example, in Sambrook, et al. (1989) and in Ausubel,et al., (Current Protocols in Molecular Biology, John Wiley & Sons, NewYork, N.Y., 1995, incorporated herein by reference).

A variety of expression vector/host systems may be utilized to containand express sequences encoding antibodies. These include, but are notlimited to, microorganisms, such as bacteria (e.g. E. coli) transformedwith recombinant bacteriophage, plasmid, or cosmid DNA expressionvectors; yeast transformed with yeast expression vectors; insect cellsystems infected with virus expression vectors (e.g., baculovirus);plant cell systems transformed with virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV); or bacterialexpression vectors (e.g., Ti or pBR322 plasmids), or animal cellsystems.

The control elements or regulatory sequences are those non-translatedregions of the vector—enhancers, promoters, 5′ and 3′ untranslatedregions—which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in strength andspecificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters such as the hybridlacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.),pSPORT1 plasmid (Life Technologies), or the like can be used. Thebaculovirus polyhedrin promoter may be used in insect cells. Promotersor enhancers derived from the genomes of plant cells (e.g., heat shock,RUBISCO, and storage protein genes) or from plant viruses (e.g., viralpromoters or leader sequences) may be cloned into the vector. Inmammalian cell systems, promoters from mammalian genes or from mammalianviruses may be used. If it is necessary to generate a cell line thatcontains multiple copies of a nucleotide sequence encoding an antibody,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

General texts describing additional molecular biological techniquesuseful herein, including the preparation of antibodies include Bergerand Kimmel (Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al.,(Molecular Cloning: A Laboratory Manual, (Second Edition, Cold SpringHarbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3);Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.],Current Protocols, a joint venture between Green Publishing Associates,Inc. and John Wiley & Sons, Inc. (supplemented through 2000)); Harlow etal., (Monoclonal Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press (1988), Paul [Ed.]); Fundamental Immunology,(Lippincott Williams & Wilkins (1998)); and Harlow, et al., (UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press(1998)), all of which are incorporated herein by reference.

Use of the dAbs of the Invention

In yet another embodiment, the present invention provides apharmaceutical composition comprising an effective amount of the domainantibody of the invention, together with a pharmaceutically acceptablecarrier or diluent.

In a particular embodiment, the present invention provides a method fortreating and/or preventing an HIV infection by administering aneffective amount of the domain antibody, m36 (or derivative thereof) ofthe invention, together with a pharmaceutically acceptable carrier ordiluent. Administration can occur before or after HIV infection.

Some terms relating to the use of the dAbs of this invention are definedas follows.

The term “treatment” includes any process, action, application, therapy,or the like, wherein a subject (or patient), including a human being, isprovided medical aid with the object of improving the subject'scondition, directly or indirectly, or slowing the progression of acondition or disorder in the subject, or ameliorating at least onesymptom of the disease or disorder under treatment.

The term “combination therapy” or “co-therapy” means the administrationof two or more therapeutic agents to treat a disease, condition, and/ordisorder. Such administration encompasses “co-administration” of two ormore therapeutic agents in a substantially simultaneous manner, such asin a single capsule having a fixed ratio of active ingredients or inmultiple, separate capsules for each inhibitor agent. In addition, suchadministration encompasses use of each type of therapeutic agent in asequential manner. The order of administration of two or moresequentially co-administered therapeutic agents is not limited.

The phrase “therapeutically effective amount” means the amount of eachagent administered that will achieve the goal of improvement in adisease, condition, and/or disorder severity, and/or symptom thereof,while avoiding or minimizing adverse side effects associated with thegiven therapeutic treatment.

The term “pharmaceutically acceptable” means that the subject item isappropriate for use in a pharmaceutical product.

The antibodies of this invention are expected to be valuable astherapeutic agents, e.g. anti-HIV antibody based therapies. Accordingly,an embodiment of this invention includes a method of treating and/orpreventing a particular condition (e.g. HIV infection) in a patientwhich comprises administering to said patient a composition containingan amount of an antibody of the invention that is effective in treatingthe target condition.

The present invention can be used to screen and identify dAb that can beused in the treatment or prevention of a variety of diseases and/orconditions, which include, for example, cancer, such as, carcinomas ofthe kidney, esophagus, breast, cervix, colon, and lung, and which alsoincludes viral infections (e.g. HIV), bacterial infections, and fungalinfections.

The domain antibodies of the present invention may be administered aloneor in combination with one or more additional therapeutic agents.Combination therapy includes administration of a single pharmaceuticaldosage formulation which contains an antibody of the present inventionand one or more additional therapeutic agents, as well as administrationof the antibody of the present invention and each additional therapeuticagents in its own separate pharmaceutical dosage formulation. Forexample, an antibody of the present invention and a therapeutic agentmay be administered to the patient together in a single oral dosagecomposition or each agent may be administered in separate oral dosageformulations.

Where separate dosage formulations are used, the antibody of the presentinvention and one or more additional therapeutic agents may beadministered at essentially the same time (e.g., concurrently) or atseparately staggered times (e.g., sequentially). The order ofadministration of the agents is not limited.

For example, in one aspect, co-administration of a domain antibody orantibody fragment of the invention together with one or more anti-HIVagents to potentiate the effect of either the antibody or the anti-HIVagent(s) or both is contemplated for use in treating HIV infections.

The one or more anti-cancer agents can include any known and suitablecompound in the art, such as, for example, chemoagents, otherimmunotherapeutics, cancer vaccines, anti-angiogenic agents, cytokines,hormone therapies, gene therapies, and radiotherapies. A chemoagent (or“anti-cancer agent” or “anti-tumor agent” or “cancer therapeutic”)refers to any molecule or compound that assists in the treatment of acancer. Examples of chemoagents contemplated by the present inventioninclude, but are not limited to, cytosine arabinoside, taxoids (e.g.,paclitaxel, docetaxel), anti-tubulin agents (e.g., paclitaxel,docetaxel, epothilone B, or its analogues), macrolides (e.g., rhizoxin)cisplatin, carboplatin, adriamycin, tenoposide, mitozantron,discodermolide, eleutherobine, 2-chlorodeoxyadenosine, alkylating agents(e.g., cyclophosphamide, mechlorethamine, thioepa, chlorambucil,melphalan, carmustine (BSNU), lomustine (CCNU), cyclothosphamide,busulfan, dibromomannitol, streptozotocin, mitomycin C, andcis-dichlorodiamine platinum (II) (DDP) cisplatin, thio-tepa),antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,mithramycin, anthramycin), antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, flavopiridol,5-fluorouracil, fludarabine, gemcitabine, dacarbazine, temozolamide),asparaginase, Bacillus Calmette and Guerin, diphtheria toxin,hexamethylmelamine, hydroxyurea, LYSODREN®, nucleoside analogues, plantalkaloids (e.g., Taxol, paclitaxel, camptothecin, topotecan, irinotecan(CAMPTOSAR, CPT-11), vincristine, vinca alkyloids such as vinblastine),podophyllotoxin (including derivatives such as epipodophyllotoxin, VP-16(etoposide), VM-26 (teniposide)), cytochalasin B, colchine, gramicidinD, ethidium bromide, emetine, mitomycin, procarbazine, mechlorethamine,anthracyclines (e.g., daunorubicin (formerly daunomycin), doxorubicin,doxorubicin liposomal), dihydroxyanthracindione, mitoxantrone,mithramycin, actinomycin D, procaine, tetracaine, lidocaine,propranolol, puromycin, anti-mitotic agents, abrin, ricin A, pseudomonasexotoxin, nerve growth factor, platelet derived growth factor, tissueplasminogen activator, aldesleukin, allutamine, anastrozle,bicalutamide, biaomycin, busulfan, capecitabine, carboplain,chlorabusil, cladribine, cylarabine, daclinomycin, estramusine,floxuridhe, gamcitabine, gosereine, idarubicin, itosfamide, lauprolideacetate, levamisole, lomusline, mechlorethamine, magestrol, acetate,mercaptopurino, mesna, mitolanc, pegaspergase, pentoslatin, picamycin,riuxlmab, campath-1, straplozocin, thioguanine, tretinoin, vinorelbine,or any fragments, family members, or derivatives thereof, includingpharmaceutically acceptable salts thereof. Compositions comprising oneor more chemoagents (e.g., FLAG, CHOP) are also contemplated by thepresent invention. FLAG comprises fludarabine, cytosine arabinoside(Ara-C) and G-CSF. CHOP comprises cyclophosphamide, vincristine,doxorubicin, and prednisone.

The chemoagent can be an anti-angiogenic agent, such as, for example,angiostatin, bevacizumab (Avastin®), sorafenib (Nexavar®), baculostatin,canstatin, maspin, anti-VEGF antibodies or peptides, anti-placentalgrowth factor antibodies or peptides, anti-Flk-1 antibodies, anti-Flt-1antibodies or peptides, laminin peptides, fibronectin peptides,plasminogen activator inhibitors, tissue metalloproteinase inhibitors,interferons, interleukin 12, IP-10, Gro-β, thrombospondin,2-methoxyoestradiol, proliferin-related protein, carboxiamidotriazole,CM101, Marimastat, pentosan polysulphate, angiopoietin 2,interferon-alpha, herbimycin A, PNU145156E, 16K prolactin fragment,Linomide, thalidomide, pentoxifylline, genistein, TNP-470, endostatin,paclitaxel, accutin, cidofovir, vincristine, bleomycin, AGM-1470,platelet factor 4 or minocycline. Without being bound by theory, thecoadministration of an anti-angiogenic agent advantageously may lead tothe increase in MN expression in a tumor, thereby making the tumor moresusceptible to the antibodies and antibody conjugates of the invention.

In one aspect, said chemoagent is gemcitabine at a dose ranging from 100to 1000 mg/m²/cycle. In one embodiment, said chemoagent is dacarbazineat a dose ranging from 200 to 4000 mg/m² cycle. In another aspect, saiddose ranges from 700 to 1000 mg/m²/cycle. In yet another aspect, saidchemoagent is fludarabine at a dose ranging from 25 to 50 mg/m²/cycle.In another aspect, said chemoagent is cytosine arabinoside (Ara-C) at adose ranging from 200 to 2000 mg/m²/cycle. In still another aspect, saidchemoagent is docetaxel at a dose ranging from 1.5 to 7.5 mg/kg/cycle.In yet another aspect, said chemoagent is paclitaxel at a dose rangingfrom 5 to 15 mg/kg/cycle. In a further aspect, said chemoagent iscisplatin at a dose ranging from 5 to 20 mg/kg/cycle. In a still furtheraspect, said chemoagent is 5-fluorouracil at a dose ranging from 5 to 20mg/kg/cycle. In another aspect, said chemo agent is doxorubicin at adose ranging from 2 to 8 mg/kg/cycle. In yet a further aspect, saidchemoagent is epipodophyllotoxin at a dose ranging from 40 to 160mg/kg/cycle. In yet another aspect, said chemoagent is cyclophosphamideat a dose ranging from 50 to 200 mg/kg/cycle. In a further aspect, saidchemoagent is irinotecan at a dose ranging from 50 to 150 mg/m²/cycle.In a still further aspect, said chemoagent is vinblastine at a doseranging from 3.7 to 18.5 mg/m²/cycle. In another aspect, said chemoagentis vincristine at a dose ranging from 0.7 to 2 mg/m²/cycle. In oneaspect, said chemoagent is methotrexate at a dose ranging from 3.3 to1000 mg/m²/cycle.

In another aspect, the anti-MN antibodies and/or antibody fragments ofthe present invention are administered in combination with one or moreimmunotherapeutic agents, such as antibodies or immunomodulators, whichinclude, but are not limited to, HERCEPTIN®, RETUXAN®, OvaRex, Panorex,BEC2, IMC-C225, Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart ID10, and Oncolym, rituxan, rituximab, gemtuzumab, or trastuzumab.

The invention also contemplates administering the domain antibodiesand/or antibody fragments of the present invention with one or moreanti-angiogenic agents, which include, but are not limited to,angiostatin, thalidomide, kringle 5, endostatin, Serpin (Serine ProteaseInhibitor) anti-thrombin, 29 kDa N-terminal and a 40 kDa C-terminalproteolytic fragments of fibronectin, 16 kDa proteolytic fragment ofprolactin, 7.8 kDa proteolytic fragment of platelet factor-4, a β-aminoacid peptide corresponding to a fragment of platelet factor-4 (Maione etal., 1990, Cancer Res. 51:2077), a 14-amino acid peptide correspondingto a fragment of collagen I (Tolma et al., 1993, J. Cell Biol. 122:497),a 19 amino acid peptide corresponding to a fragment of Thrombospondin I(Tolsma et al., 1993, J. Cell Biol. 122:497), a 20-amino acid peptidecorresponding to a fragment of SPARC (Sage et al., 1995, J. Cell.Biochem. 57:1329-), or any fragments, family members, or derivativesthereof, including pharmaceutically acceptable salts thereof.

Other peptides that inhibit angiogenesis and correspond to fragments oflaminin, fibronectin, procollagen, and EGF have also been described (Seethe review by Cao, 1998, Prog. Mol. Subcell. Biol. 20:161). Monoclonalantibodies and cyclic pentapeptides, which block certain integrins thatbind RGD proteins (i.e., possess the peptide motif Arg-Gly-Asp), havebeen demonstrated to have anti-vascularization activities (Brooks etal., 1994, Science 264:569; Hammes et al., 1996, Nature Medicine 2:529).Moreover, inhibition of the urokinase plasminogen activator receptor byantagonists inhibits angiogenesis, tumor growth and metastasis (Min etal., 1996, Cancer Res. 56:2428-33; Crowley et al., 1993, Proc Natl Acad.Sci. USA 90:5021). Use of such anti-angiogenic agents is alsocontemplated by the present invention.

In another aspect, the domain antibodies and/or antibody fragments ofthe present invention are administered in combination with a regimen ofradiation.

The domain antibodies and/or antibody fragments of the present inventioncan also be administered in combination with one or more cytokines,which includes, but is not limited to, lymphokines, tumor necrosisfactors, tumor necrosis factor-like cytokines, lymphotoxin-α,lymphotoxin-β, interferon-β, macrophage inflammatory proteins,granulocyte monocyte colony stimulating factor, interleukins (including,but not limited to, interleukin-1, interleukin-2, interleukin-6,interleukin-12, interleukin-15, interleukin-18), OX40, CD27, CD30, CD40or CD137 ligands, Fas-Pas ligand, 4-1BBL, endothelial monocyteactivating protein or any fragments, family members, or derivativesthereof, including pharmaceutically acceptable salts thereof.

The domain antibodies and/or antibody fragments s of the presentinvention can also be administered in combination with a cancer vaccine,examples of which include, but are not limited to, autologous cells ortissues, non-autologous cells or tissues, carcinoembryonic antigen,alpha-fetoprotein, human chorionic gonadotropin, BCG live vaccine,melanocyte lineage proteins (e.g., gp100, MART-1/MelanA, TRP-1 (gp75),tyrosinase, widely shared tumor-associated, including tumor-specific,antigens (e.g., BAGE, GAGE-1, GAGE-2, MAGE-1, MAGE-3,N-acetylglucosaminyltransferase-V, p15), mutated antigens that aretumor-associated (β-catenin, MUM-1, CDK4), nonmelanoma antigens (e.g.,HER-2/neu (breast and ovarian carcinoma), human papillomavirus-E6, E7(cervical carcinoma), MUC-1 (breast, ovarian and pancreatic carcinoma).For human tumor antigens recognized by T-cells, see generally Robbinsand Kawakami, 1996, Curr. Opin. Immunol. 8:628. Cancer vaccines may ormay not be purified preparations.

In yet another embodiment, the domain antibodies and/or antibodyfragments of the present invention are used in association with ahormonal treatment. Hormonal therapeutic treatments comprise hormonalagonists, hormonal antagonists (e.g., flutamide, tamoxifen, leuprolideacetate (LUPRON), LH-RH antagonists), inhibitors of hormone biosynthesisand processing, and steroids (e.g., dexamethasone, retinoids,betamethasone, cortisol, cortisone, prednisone, dehydrotestosterone,glucocorticoids, mineralocorticoids, estrogen, testosterone,progestins), antigestagens (e.g., mifepristone, onapristone), andantiandrogens (e.g., cyproterone acetate).

The antibodies described herein may be provided in a pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier. Thepharmaceutically acceptable carrier may be non-pyrogenic. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier including, but notlimited to, saline, buffered saline, dextrose, and water. A variety ofaqueous carriers may be employed including, but not limited to saline,glycine, or the like. These solutions are sterile and generally free ofparticulate matter. These solutions may be sterilized by conventional,well-known sterilization techniques (e.g., filtration).

Generally, the phrase “pharmaceutically acceptable carrier” is artrecognized and includes a pharmaceutically acceptable material,composition or vehicle, suitable for administering compounds of thepresent invention to mammals. The carriers include liquid or solidfiller, diluent, excipient, solvent or encapsulating material, involvedin carrying or transporting the subject agent from one organ, or portionof the body, to another organ, or portion of the body. Each carrier mustbe “acceptable” in the sense of being compatible with the otheringredients of the formulation and not injurious to the patient. Someexamples of materials which can serve as pharmaceutically acceptablecarriers include: sugars, such as lactose, glucose and sucrose;starches, such as corn starch and potato starch; cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients,such as cocoa butter and suppository waxes; oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; glycols, such as propylene glycol; polyols, such asglycerin, sorbitol, mannitol and polyethylene glycol; esters, such asethyl oleate and ethyl laurate; agar; buffering agents, such asmagnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-freewater; isotonic saline; Ringer's solution; ethyl alcohol; phosphatebuffer solutions; and other non-toxic compatible substances employed inpharmaceutical formulations.

Wetting agents, emulsifiers and lubricants, such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releaseagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the antibodycompositions of the invention.

Examples of pharmaceutically acceptable antioxidants include: watersoluble antioxidants, such as ascorbic acid, cysteine hydrochloride,sodium bisulfate, sodium metabisulfite, sodium sulfite and the like;oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

The compositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions such aspH adjusting and buffering agents, and the like. The concentration ofthe antibody of the invention in such pharmaceutical formulation mayvary widely, and may be selected primarily based on fluid volumes,viscosities, etc., according to the particular mode of administrationselected. If desired, more than one type of antibody may be included ina pharmaceutical composition (e.g., an antibody with different K_(d) forMN binding).

The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones. In addition to theactive ingredients, these pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries that facilitate processing of the active compounds intopreparations which may be used pharmaceutically. Pharmaceuticalcompositions of the invention may be administered by any number ofroutes including, but not limited to, oral, intravenous, intramuscular,intra-arterial, intramedullary, intrathecal, intraventricular,transdermal, subcutaneous, intraperitoneal, intranasal, parenteral,topical, sublingual, or rectal means.

The compositions of the invention additionally contemplate suitableimmunocarriers, such as, proteins, polypeptides or peptides such asalbumin, hemocyanin, thyroglobulin and derivatives thereof, particularlybovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH),polysaccharides, carbohydrates, polymers, and solid phases. Otherprotein-derived or non-protein derived substances are known to thoseskilled in the art.

Formulations suitable for parenteral, subcutaneous, intravenous,intramuscular, and the like; suitable pharmaceutical carriers; andtechniques for formulation and administration may be prepared by any ofthe methods well known in the art (see, e.g., Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 20^(th) edition, 2000).Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluent commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular,subarachnoid, intraspinal and intrasternal injection and infusion.

The determination of a therapeutically effective dose is well within thecapability of those skilled in the art. A therapeutically effective doserefers to the amount of an antibody that may be used to effectivelytreat a disease (e.g., cancer) compared with the efficacy that isevident in the absence of the therapeutically effective dose.

The therapeutically effective dose may be estimated initially in animalmodels (e.g., rats, mice, rabbits, dogs, or pigs). The animal model mayalso be used to determine the appropriate concentration range and routeof administration. Such information may then be used to determine usefuldoses and routes for administration in humans.

Therapeutic efficacy and toxicity (e.g., ED₅₀—the dose therapeuticallyeffective in 50% of the population and LD₅₀—the dose lethal to 50% ofthe population) of an antibody may be determined by standardpharmaceutical procedures in cell cultures or experimental animals. Thedose ratio of toxic to therapeutic effects is the therapeutic index, andit may be expressed as the ratio, LD₅₀/ED₅₀. The data obtained fromanimal studies may used in formulating a range of dosage for human use.The dosage contained in such compositions may be within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage may be determined by the practitioner, in light offactors related to the patient who requires treatment. Dosage andadministration may be adjusted to provide sufficient levels of theantibody or to maintain the desired effect. Factors that may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy.

The antibodies of the invention may also be administered by introducinggenetically engineered bacteria which express and release the domainantibodies of the invention once the bacteria a present in the patient.This format might be suitable for treating HIV infections. Theantibody-expressing bacteria can be introduced into mucus membranes ofthe throat, for example, or in other mucosal regions in which HIV mightbe found. Methods for constructing and/or engineering such recombinantbacteria are well known in the art.

Polynucleotides encoding antibodies of the invention may be constructedand introduced into a cell either ex vivo or in vivo usingwell-established techniques including, but not limited to,transferrin-polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated cellular fusion,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, “gene gun,” and DEAE- orcalcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 μgto about 500 μg/kg of patient body weight. For administration ofpolynucleotides encoding the antibodies, effective in vivo dosages arein the range of about 100 ng to about 500 μg of DNA.

The antibodies of the present invention can also be delivered in amicrosphere or microsome bodies.

The mode of administration of antibody-containing pharmaceuticalcompositions of the present invention may be any suitable route whichdelivers the antibody to the host. As an example, pharmaceuticalcompositions of the invention may be useful for parenteraladministration (e.g., subcutaneous, intramuscular, intravenous, orintranasal administration, or microsomal or lipid microsome bodies).

The above disclosure generally describes the present invention. A morecomplete understanding can be obtained by reference to the followingspecific examples, which are provided for purposes of illustration onlyand are not intended to limit the scope of the invention.

EXAMPLES

The structures, materials, compositions, and methods described hereinare intended to be representative examples of the invention, and it willbe understood that the scope of the invention is not limited by thescope of the examples. Those skilled in the art will recognize that theinvention may be practiced with variations on the disclosed structures,materials, compositions and methods, and such variations are regarded aswithin the ambit of the invention.

Example 1 Identification of VH Framework

A naïve human Fab library (1.5×10¹⁰ members) was constructed fromperipheral blood B cells of 22 healthy donors, spleens of 3 donors, andlymph nodes of healthy 34 donors. This library was used for selection ofFabs against HIV-1 antigen gp140 which was conjugated to magnetic beads(Dynabeads M-270 epoxy; DYNAL Inc., New Hyde Park, N.Y.) as described(Zhu Z et al. J Viol. 2006, 80(2):891-9). Amplified libraries of3.8×10¹² phage-displayed Fabs were incubated with 20, 20, 10, and 10 μgof gp140 in 1 ml volume for 2 h at room temperature during the first,second, third, and fourth rounds of biopanning, respectively. Afterincubation the beads were washed 5 times for the first round and 10times for the later rounds with PBST to remove nonspecifically boundphages. Bound phages were rescued by mixing the beads with E. coli TG1cells for 45 min at 37° C. and a phage library was prepared for the nextround of biopanning. Ninety five clones were randomly picked from theinfected TG1 cells in the third and fourth round, respectively, andsubjected to monoclonal phage-based enzyme-linked immunosorbent assay(monoclonal phage ELISA). A positive Fab, designated R3H1, wasidentified with a stop codon in the light chain. Its reading frame wascorrected subsequently. R3H1 was expressed and purified as shown in FIG.4.

The gene fragment of R3H1 VH domain (m0) was amplified from R3H1Fabphagemid and subcloned into phagemid vector (e.g. pComb3x or pZYD). Thephagemids containing m0 genes were prepared and transformed to E. coliHB2151 chemical competent cells. Soluble m0 were expressed and purifiedas described (Zhu Z et al. J Virol. 2006, 80(2):891-9). The SDS-PAGE ofpurified m0 was shown in FIG. 4. The amino acid and nucleotide sequencesof m0 are shown in FIG. 5.

Example 2 Construction and Screening of Domain Antibody Library

Highly diverse antibody libraries have become important sources forselection of antibodies with high affinity and novel properties.Combinatorial strategies provide efficient ways of creating antibodylibraries containing a large number of individual clones. Thesestrategies include the reassembly of naturally occurring genes encodingthe heavy and light chains from either immune or nonimmune B-cellsources and introduction of synthetic diversity to either the frameworkregions (FRs) or the complementarity determining regions (CDRs) of thevariable domains of antibodies.

This Example describes the identification of a human heavy chain-onlyantibody and its use as a scaffold for construction of a phage-displayedVH library as well as an approach to introduce genetic diversity in thislibrary in which natural human CDR2, CDR3, and synthetic CDR1repertoires are combined into a single human VH framework scaffold. Theusefulness of the library has been demonstrated by the successfulselection of high-affinity binders to viral and cancer-related antigens.

The following methods and materials were employed in this Example. Theresults of this Example are subsequently discussed below.

Methods and Materials:

Amplification of CDR Repertoire and FR Fragments

Primers used for PCR amplification of gene fragments are described inTable 1, below.

TABLE 1 Primers used for PCR amplification of gene fragments. Primerdescription Name Sequence Target H1 antisense H1R 5′-GCG GAC CDR1CCA GCT CAT TTC ATA AKM AKM GAA AKM GAA AKM AGA GGC TGC ACA GGA GAG(SEQ ID NO: 5)^(c) H1 sense H1-F 5′-GAG GAG CDR1 GAG GAG GAG GAG GCG GGGCCC AGG CGG CCC AGG TGC AGC TGG TGC- 3′ (SEQ ID NO: 6) H2 sense H2F15′-GAA ATG VH1-2^(a), VH1-3, AGC TGG GTC VH1-8, VH1-18, CGC CAG GCTVH1-45, VH1-46, CCA GGA CAA VH1-58, VH1-69, SGS CTT GAG VH1-C, VH6-1,TGG VH7-*^(b) (SEQ ID NO: 7) H2F2 GAA ATG AGC VH2-* TGG GTC CGCCAG GCT CCA GGG AAG GCC CTG GAG TGG (SEQ ID NO: 8) H2F3 5′-GAA ATGVH1-24, VH1-F, AGC TGG GTC VH3-*, VH4-*, CGC CAG GCT VH5-* CCA GGG AAGGGN CTR GAG TGG (SEQ ID NO: 9) H2 antisense H2R1 ATT GTC TCT VH1-*GGA GAT GGT GAC CCT KYC CTG RAA CTY (SEQ ID NO: 10) H2R2 5′-ATT GTC TCT VH3-*, VH6-1 GGA GAT GGT GAA TCG GCC CTT CAC NGA (SEQ ID NO: 11) H2R35′-ATT GTC TCT  VH2-*, VH4-* GGA GAT GGT GAC TMG ACT CTT GAG GGA(SEQ ID NO: 12) H2R4 5′-ATT GTC TCT  VH5-* GGA GAT GGT GAC STG GCCTTG GAA GGA (SEQ ID NO: 13) H2R5 5′-ATT GTC TCT  VH7-* GGA GAT GGTAAA CCG TCC TGT GAA GCC (SEQ ID NO: 14) H3 sense H3F1 5′-ACC CTGVH3-9, VH3-20, AGA GCC GAG VH3-43 GAC ACR GCY TTR TAT TAC TGT(SEQ ID NO: 15) H3F2 5′-ACC CTG VH1-45, VH2-*, AGA GCC GAG VH5-*, VH7-81GAC ACA GCC AYR TAT TAC TGT (SEQ ID NO: 16) H3F3 5′-ACC CTGOther than above AGA GCC GAG GAC ACR GCY GTR TAT TAC TGT (SEQ ID NO: 17)H3 antisense H3R 5′-GTG GCC FR4 GGC CTG GCC ACT TGA GGA GAC GGT GAC C(SEQ ID NO: 18) FR3 sense FR3F 5′-ACC ATC TCC  FR3 AGA GAC AAT TCC(SEQ ID NO: 19) FR3 antisense FR3R 5′-GTC CTC GGC  FR3 TCT CAG GGT G(SEQ ID NO: 20) Extension #1 FR1F 5′-TGG TTT CGC  FR1 TAC CGT GGCCCA GGC GGC CCA GGT GCA GCT GGT G (SEQ ID NO: 21) Extension #2 H1SR5′-GTC GCC GTG  5′ end of H3R GTG GTG GTG primer GTG GTG GCC GGC CTG GCCACT TG (SEQ ID NO: 22) ^(a)Sub-groups of human antibody heavy chaingenes. ^(b)All members in the groups. ^(c)Underlined codons indicatesprimer degeneracy.

For amplification of CDR1 repertoire and FR3 fragment, the master humanVH m0, as described in Example 1 and shown in FIG. 5, was used as thetemplate. The degenerate primer H1R (see Table 1) enables randomizationof four amino acids in CDR1 to A/D/S/Y. Three non-immune (IgM-derivedfrom healthy non-infected non-immunized donors) human antibody phagedisplay libraries that have recently been constructed according to theprocedure described previously (de Haard H J et al. 1999. J. Biol. Chem.274:18218-30) and one immune (IgG-derived from HIV-infected donors)library constructed from HIV patients have been used as templates foramplification of CDR2 and CDR3 repertoires (See FIG. 6A). The librariesused included (a) a naïve human Fab library (5×10⁹ members) constructedfrom peripheral blood B cells of 10 healthy donors (Zhu et al., J.Virol., 2006, which is incorporated herein by reference); (b) a naïvehuman Fab library (1.5×10¹⁰ members) constructed from peripheral blood Bcells of 22 healthy donors, spleens of 3 donors, and lymph nodes ofhealthy 34 donors (de Haard H J et al. 1999. J. Biol. Chem.274:18218-30); (c) a naïve human Fab library (7.2×10⁸ members)constructed from cord blood of 2 healthy babies (essentially theprocedure and primers described in (de Haard H J et al. 1999. J. Biol.Chem. 274:18218-30) were used but with one-step overlapping PCR cloning(Zhu Z and Dimitrov D S, in press, Therapeutic antibodies, A. Dimitrov,Ed., Methods in Molecular Biology, Humana Press; and (d) an immune humanFab library constructed from bone marrow obtained from 3 long-termnonprogressors whose sera exhibited the broadest and most potent HIV-1neutralization among 37 HIV-infected individuals (Zhang et al., J.Immunol. Methods, 2003). To maintain maximal diversity, 8 first roundPCR reactions were carried out using different primer combinations ofTable 2 against each of the above libraries as a template to obtain CDR2product repertoire. Similarly, 3 first round PCR reactions were carriedout using different primer combinations of Table 2 against each of theabove libraries as a template to obtain CDR3 product repertoire.

TABLE 2 Primer pairings used for first round amplification of genesegments Primer pairings Products Targets FR1F-H1R FR1-CDR1-FR2^(a) CDR1H2F1-H2R1 FR2^(a)-CDR2-FR3^(a) VH1-* (except VH1-24, VH1-F) H2F1-H2R2FR2^(a)-CDR2-FR3^(a) VH6-1 H2F1-H2R5 FR2^(a)-CDR2-FR3^(a) VH7-*H2F2-H2R3 FR2^(a)-CDR2-FR3^(a) VH2-* H2F3-H2R1 FR2^(a)-CDR2-FR3^(a)VH1-24, VH1-F H2F3-H2R2 FR2^(a)-CDR2-FR3^(a) VH3-* H2F3-H2R3FR2^(a)-CDR2-FR3^(a) VH4-* H2F3-H2R4 FR2^(a)-CDR2-FR3^(a) VH5-* H3F1-H3RFR3^(a)-CDR3-FR4 VH3-9, VH3-20, VH3-43 H3F1-H3F2 FR3^(a)-CDR3-FR4VH1-45, VH2-*, VH5-*, VH7-81 H3F1-H3F3 FR3^(a)-CDR3-FR4 Other than aboveFR3F-FR3R FR3 FR3 ^(a)These products partially cover FRs.

PCRs were performed in a volume of 50 μl using High Fidelity PCR Master(Roche, Cat. #12140314001), 500 pM of each primer and 0.5 μg of templateDNA (e.g. VH m0 or the four antibody phage display libraries describedabove) for 30 cycles (45 sec at 94° C.; 45 sec at 55° C.; and 1 min at72° C.). Products for each primer combination from one template werepooled, purified from 2% agarose gel using QIAquick Gel Extraction Kit(Qiagen, Cat. #28706), and quantified by reading the optical density(O.D.) at 260 nm (1 O.D. unit=50 μg/ml). Finally, all products from thefour antibody phage display library templates were pooled at a molarityratio calculated by counting the number of donors for templatelibraries, e.g. 10:59:2:3.

Assembly of Complete VH Domain Antibodies

The primers used in the first round of PCR created identical sequencesin the downstream regions of the CDR1 products, the upstream regions ofCDR3 products, and both the downstream and the upstream regions of theCDR2 products. These identical sequences are homologous to FRs and serveas the overlap for the second- and third-round extension PCR reactions.

In the second-round PCR extension reactions (see FIG. 6B), CDR1fragments containing the whole FR1 and partial FR2 on both sides werejoined to the CDR2 fragments containing partial FR2 and partial FR3 onboth sides by overlapping PCRs performed in a volume of 100 μl usingboth templates (in the same molarities) for 7 cycles in the absence ofprimers and additional 15 cycles in the presence of primers (500 pM ofFR1F and 500 pM of H2R1-5 mixture). Under the same condition, FR3fragments were joined to CDR3 fragments containing partial FR3 and thewhole FR4 on both sides by overlapping PCRs using primers FR3F and H3R.

In the third-round extension (see FIG. 6C), complete VHs were formed byannealing the products of the second-round extension reactions to eachother using overlapping PCRs with the extension primers HISR and FR1Fappended with SfiI restriction sites.

Preparation of VH Domain Library

Gel-purified VH products formed above were digested with SfiI (NewEngland BioLabs, Inc., Cat. #R0123L), and cloned into a phagemid vector.The SfiI-digested and gel-purified VH fragments and phagemid vectors, 80μg and 230 μg respectively, were ligated in an 8-ml reaction mixturewith 10000 units of T4-DNA ligase (New England BioLabs, Inc., Cat.#M0202L) at 16° C. for 72 h. The ligation products were then desaltedand concentrated by passing through a 4-ml Amicon Ultra-4 centrifugalfilter with a cutoff of 3000 MW (Millipore, Cat. #UFC800324) at 4000×gfor 20 minutes at room temperature and washing 3 times with 4 ml ofdistilled water. About 100-μl of ligation product was recovered from thefilter and stored at −20° C. for later use.

Electroporation was then performed. For electroporations, 1 L of 2YTmedium containing 1% glucose (w/v) was pre-warmed at 37° C. 100 genepulser cuvettes (Bio-Rad, Cat. #165-2089) with 1 mm gap were chilled onice. The desalted ligation product and 4 ml of E. coli strain TG1electroporation-competent cells (Stratagene, Cat. #200123) were thawedtogether on ice. The TG1 competent cells were divided into 10pre-chilled 1.5-ml Eppendorf tubes in 400 μl aliquots. Ten μl ofligation product was added to each tube and gently mixed by pipetteaction. Next, 41 μl of mixture was transferred to each electroporationcuvette, which was gently tapped on the bench to move the mixture to thebottom of the cuvette. Electroporations were performed at 1.8 kV, 25 μF,and 200Ω and the cuvettes were flushed immediately with 1 ml and thentwice with 2 ml of pre-warmed 2YT medium and transferred into a 2-Lflask.

After completing all electroporations, 700 ml of pre-warmed 2YT mediumwas added to each flask to make a volume of 1 L in total. The cultureswere incubated at 37° C. with shaking at 250 rpm for 30 min. Ten μl ofthe culture was 10-fold serially diluted in 100 μl of 2YT medium, andplated on 2YT agar plates containing 2% glucose (w/v) and 100 μg/ml ofampicillin. The plates were incubated overnight at 37° C. The totalnumber of transformants was calculated by counting the number ofcolonies, multiplying by the culture volume, and dividing by the platingvolume.

For preparation of the phage display library, 1 ml of 100 mg/mlampicillin was added to the 1-L culture and the culture was thenincubated with shaking for additional 2 h at 37° C. The cell density wasmeasured by reading the O.D.600 of the culture and the total number ofcells was calculated by multiplying the O.D.600 value by 5×10⁸(estimated number of cells in 1 ml culture when O.D.600 reaches 1) andthe culture volume (1000 in this case). The culture was infected with 10M.O.I. of M13KO7 helper phage (New England BioLabs, Cat. #N0315S) andincubated at 37° C. for 30 min, shaking for homogenization every 10 min.The cells were collected by centrifuging at 5000 rpm for 10 min andresuspended in 2-L 2YT medium containing 100 μg/ml of ampicillin and 50μg/ml of kanamycin. Following incubation at 250 rpm overnight at 30° C.,the culture was centrifuged at 5000 rpm for 15 min at 4° C.

The phagemids were prepared from the bacterial pellet using the QiagenHiSpeed Plasmid Maxi Kit (Cat. #12663). For phage precipitation, thesupernatant was transferred to 2 clean 2-L flasks, mixed well with ¼volume of PEG8000 (20%, w/v)/NaCl (2.5 M) solution, and incubated on icefor 3 h. The mixture was centrifuged at 14000×g for 20 min at 4° C. andthe phage pellet was resuspended in 100 ml 1×PBS, pH7.4 by pipetting upand down along the side of the centrifuge bottles with a 10-ml pipette.The phage suspension was centrifuged at 5000 rpm for 10 min at 4° C. Thesupernatant was transferred to a clean 200 ml flask, mixed well with ¼volume of PEG8000 (20%, w/v)/NaCl (2.5 M) solution, and incubated on icefor 1 h. The mixture was centrifuged at 14000×g for 20 min at 4° C. andthe phage pellet was resuspended in 50 ml 1×PBS, pH7.4. The phagesuspension was centrifuged at 5000 rpm for 10 min at 4° C. and thesupernatant was transferred to a clean 200 ml flask. The concentrationof phage was measured by reading O.D.280 (1 O.D.280=2.33×10¹²/ml). Forlong-term storage, the phages were mixed with the same volume ofautoclaved glycerol, aliquoted to make sure that each contains phageparticles at least 100 times of the total number of transformants, andstored at −80° C.

Sequence Diversity Analysis

To evaluate sequence diversity of the VH domain library, clones wererandomly selected from the library and sequenced. The amino acidsequences were deduced from those clones with complete nucleotidesequences. For analysis of CDR1 sequence diversity, the frequency ofA/D/S/Y usage in each position mutated was calculated (see FIG. 8A).

The origins of CDR2s were determined and the numbers of mutations inamino acid sequences were calculated by comparing to the germlines ofhuman VHs from IMGT database(http://imgt.cines.fr/textes/IMGTrepertoire/Proteins/protein/human/IGH/IGHV/Hu_IGHVallgenes.html).The FR sequences on both sides of CDR2, e.g. residue #53-55 (IMGTnumbering) and residue #70-76, are highly diversified among 7 groups ofhuman VH germlines, thus, could be used as markers in addition to CDR2sequences themselves to determine CDR2 origins. See FIGS. 8B and 8C.

The length of CDR3 was calculated one by one and the length distributionwas compared to that of human heavy chain CDR3 in vivo. The pairingbetween CDR2 origins and lengths of CDR3 was also plotted. See FIG. 8D.

Measurement of VH Folding

The folding of phage-displayed VHs with a fixed VH3 scaffold wasmeasured by their ability binding to protein A. The use of Protein A asa marker for proper folding of human VH3 is well known. The library of˜10¹³ phage particles was blocked in 2% non-fat dry milk in PBS for 1 hat room temperature and passed through a chromatography column (Bio-Rad,Cat. #731-1550) loaded with 300 ml of nProtein A Sepharose 4 Fast Flow(GE Healthcare, Cat. #17-5280-02). The column was washed 3 times with 10ml of PBS containing 0.05% (v/v) Tween-20 (PBST) each. Bound phages wereeluted by incubation at room temperature for 10 min with 1 ml of 100 mMacetic acid (pH 3.0) followed by neutralization with 0.1 ml of 1 MTris-HCl (pH 9.0). Eluted phage were rescued by infection of E. coli TG1cells and a phage library was prepared for the next round of selection.In the fourth round of selection, TG1 cells were infected with theeluted phages, serially diluted and plated on 2YT agar plates. Cloneswere randomly selected from these plates and sequenced. The origins ofCDR2s were determined as described above.

Panning the Library to Screen for Domain Antibodies of Interest

The library of ˜10¹³ phage particles was blocked in 1 ml PBS containing2% non-fat dry milk and incubated with 10 μg of gp120-CD4, 5 μg ofgp140, 5 μg of gp120-CD4, and 5 μg of gp140 conjugated to magnetic beads(Dynabeads M-270 epoxy, Invitrogen, Cat. #143-01) for 2 h at roomtemperature during the first, second, third and fourth rounds ofbiopanning, respectively. After incubation the beads were washed 5 timesfor the first round and 15 times for the later rounds with PBST toremove nonspecifically bound phages. Bound phage were rescued by mixingthe beads with E. coli TG1 cells for 45 min at 37° C. and a phagelibrary was prepared for the next round of biopanning Clones wererandomly picked from the infected TG1 cells in the third and fourthround and subjected to monoclonal phage-based enzyme-linkedimmunosorbent assay (monoclonal phage ELISA) to identify clones of phagedisplaying VHs with binding activity as described (Zhu et al., J.Virol., 2006, which is incorporated herein by reference).

Each of the selected clones was inoculated into 100 μl of 2YT mediumcontaining 100 μg/ml ampicillin and 0.2% glucose in 96-well plates. Whenthe bacterial cultures reached an O.D. 600 of 0.5, 50 μl of fresh 2YTmedium containing helper phage M13KO7 at M.O.I. of 10 and kanamycin at50 μg/ml (final concentration) was added to each well, and the plateswere further incubated at 30° C. overnight in a shaker at 250 rpm. 50 μlof each phage supernatant was moved to a Corning high-binding 96-wellplate (Sigma, Cat. #CLS3690) coated with 1 μg/ml of gp120-CD4 andblocked with 3% non-fat dry milk in PBS, and incubated for 2 h at roomtemperature. The plates were washed 4 times with PBST and incubated with50 μl of horse radish peroxidase (HRP)-conjugated anti-M13 antibody (GEHealthcare, Cat. #27-9421-01) 1:5000 diluted in PBS at room temperaturefor 1 h. After incubation the plates were washed 4 times with PBST and50 μl of substrate solution ABST (Roche, Cat. #1684302) was added toeach well. The solution absorbance at 405 nm (A405) was measuredfollowing incubation at 37° C. for 15-30 min.

Expression and Purification of VH Domain Antibodies

The selected clones resulting from the panning and screening process,each phagemid clone containing a VH gene of interest, were prepared andtransformed into E. coli HB2151 chemical competent cells. These selectedphagemid clones were referred to as c3, c6, d1, d7, b4, c11, d10, b3, b5(also referred to herein as m36), b7, g6 and e11. Soluble VHs wereexpressed as described previously (Zhu et. al., J. Virol., 2006, whichis incorporated herein by reference). The bacterial pellet was collectedafter centrifugation at 8000×g for 10 min and resuspended in PBS buffercontaining 0.5 mU polymixin B (Sigma, Cat. #P0972). After 30 minincubation with rotation at 50 rpm at room temperature, it wascentrifuged at 25000×g for 25 min at 4° C., and the supernatant was usedfor Hexahistidine-tagged VH purification by immobilized metal ionaffinity chromatography (IMAC) using Ni-NTA resin (Qiagen, Cat. #30230)according to manufacturer's protocols (which are incorporated herein byreference). The purity of each VH was determined by running SDS-PAGE(see FIG. 7 or FIG. 11C) and measuring O.D. at 280.

Binding of Soluble VH Domain Antibodies

ELISA was performed by using Corning high-binding 96-well plates coatedwith 1 μg/ml of antigens and blocked with 3% non-fat dry milk in PBS.Microplate wells were then inoculated with 50 μl of serially dilutedsoluble VHs for 2 h at room temperature. After 4 washes with PBST,FLAG-tagged VHs were detected by adding 50 μl of 1:5000 dilutedHRP-conjugated anti-FLAG antibody (Sigma, Cat. #A8592) to each well.Following incubation with the antibody for 1 h at room temperature, theplates were washed 4 times with PBST and the assay was developed at 37°C. with ABST substrate and monitored at 405 nm as described.

Measurement of VH Domain Antibody Oligomerization

Superdex75 column was calibrated with protein molecular mass standard of13.7 (ribonuclease A), 25 (chymotrypsin), 44 (ovalbumin), 67 (albumin),158 (aldolase), 232 (catalase), 440 (ferritin) and 669 (thyroglobulin)kDa. Purified VH domain antibodies in PBS were loaded onto the columnthat had been pre-equilibrated with PBS. The proteins were eluted withPBS at 0.5 ml/min.

Pseudovirus Neutralization Assay

Viruses pseudotyped with Envs from HIV-1 primary isolates representingHIV-1 group M, clades A-E (4) were used in this study. Briefly,pseudotyped viruses were prepared by cotransfection of 70-80% confluent293T cells with pNL4-3.luc.E-R- and pSV7d-env plasmid using the PolyFecttransfection reagent, according to manufacturer's instruction (Qiagen,Cat. #301105). Pseudotyped viruses were obtained after 24 h bycentrifugation and filtration of cell culture through 0.45-μm filters,mixed with different concentrations of antibodies for 1 h at 37° C., andthen added to 1.5×10⁴ HOS-CD4-CCR5 cells grown in each well of 96-wellplates. Luminesence was measured after 2 days, using the Bright-GloLuciferase Assay System (Promega, Cat. #E2610) and a LumiCountmicroplate luminometer (Turner Designs). Mean relative light units (RLU)for triplicate wells were determined. Percentage inhibition wascalculated by the following formula: (1−average RLU ofantibody-containing wells/average RLU of virus-only wells)×100. IC₅₀ andIC₉₀ of neutralization were assigned for the antibody concentration atwhich 50% and 90% neutralization were observed, respectively.

A more detailed description of the specific materials that could beemployed in carrying out the methods of Example 2 are outlined asfollows. Equivalent materials can be employed as will be understood byone of ordinary skill in the art. This specific listing of materials isnot meant to limit the present invention in any way.

Detailed List of Materials:

The following list of materials were used in connection with the humanVH genes and phagemid vectors

-   1. VH framework m0 is shown in FIG. 20A.-   2. Phagemid vector is shown in FIG. 20B.

The following list of materials were used in connection with theisolation of lymphocytes.

-   1. Defibrinated or anticoagulant-treated human peripheral blood    stored at 4° C. was used as soon as possible. Total RNA, PolyA+ RNA,    and cDNA of human blood and other immune tissues such as bone    marrow, spleen, and lymph node are commercially available, e.g. from    Clontech.-   2. Ficoll-Paque Plus regents (Amersham Bioscience, Piscataway, N.J.)    were used.-   3. Solution A: 0.1% (w/v) anhydrous D-glucose, 0.05 mM CaCl₂, 0.98    mM MgCl₂, 5.4 mM KCl, and 145 mM Tris was used. To prepare, Solution    A was dissolved in approximately 950 ml double distilled water    (ddH₂O) and add 10 N HCl until pH is 7.6. The volume was adjusted to    1 L with ddH₂O.-   4. Solution B: 140 mM NaCl in ddH₂O was used.-   5. Balanced salt solution (ready to use) was employed. To prepare, 1    volume of Solution A was mixed with 9 volumes of Solution B. The    balanced salt solution should be prepared fresh each week.    Equivalent salt solution may be employed, e.g., phosphate buffer    solution (PBS), pH 7.4.-   6. Eppendorf centrifuge 5804R (Eppendorf, Westbury, N.Y.) was used    for centrifugation. Any equivalently refrigerated centrifuge    producing up to at least 400 g and maintaining temperature of    18-20° C. may be employed.-   7. BD Falcon™ Conical Tubes (BD Biosciences, San Jose, Calif.) we    used. Any equivalent tubes with a volume of ˜15 ml and internal    diameter ˜1.3 cm may be used.-   8. Pasteur pipettes, 3 ml, were used.-   9. Hemacytometer (Sigma, St. Louis, Mo.) was used.-   10. 0.4% trypan blue stain (Sigma, St. Louis, Mo.) was used.

The following list of materials were used in connection with theisolation of total RNA and the synthesis of cDNA.

-   1. RNeasy Mini Kits (Qiagen, Valencia, Calif.) were employed.-   2. QlAshredder (Qiagen, Valencia, Calif.) kits were employed.-   3. SuperScript. III First-Strand Synthesis System for RT-PCR    (Invitrogen, Carlsbad, Calif.) was employed.-   4. Corning® PCR tubes, free of RNase and DNase (Sigma, St. Louis,    Mo.) were employed.-   5. 1.5 ml Eppendorf tubes, treated with distilled water containing    0.05% (v/v) DEPC at 37° C. overnight, dried in an oven, and    autoclaved, were employed.-   6. Ultra pure water (Quality Biologicals, Gaithersburg, Md.), free    of RNase and DNase was employed.-   7. Eppendorf centrifuge 5417R (Eppendorf, Westbury, N.Y.), or other    refrigerated centrifuges with adapters for 1.5 ml Eppendorf    centrifugal tubes were employed.-   8. Bio-Rad PTC-100 thermal cycler (Bio-Rad, Hercules, Calif.) was    employed. Any equivalent thermal cycler with a hot bonnet heated lid    may be employed.

The following list of materials were used in connection with the PCRamplification of CDRs and FRs and the assembly of entire VHs.

-   1. High Fidelity PCR Master (Roche, Indianapolis, Ind.) was used.    Other high-fidelity PCR systems may be used.-   2. Primers for PCR amplification of CDRs can be found in Table 1    above. It is noted that to construct a highly diverse antibody    library, it is essential for the primers to be able to cover as many    human antibody genes as possible. To design those primers, it is    necessary to possess some sequence information and try to understand    how human antibody genes are organized. It is further noted that the    products of CDR1 primers and CDR2 primers will cover FR1 and FR2,    respectively, so that there are only four fragments instead of six    (FR1-3 and CDR1-3) for the entire VH assembly. The number of    fragments for assembly should be reduced to help decrease reading    frame shifts. It was found that highly efficient digestion of VH    products with restriction enzymes is critical for the construction    of a large library. PCR amplification using the extension primers    will result in long overhangs at both 5′ and 3′ ends of VH products    so that there is an obvious difference in length change after    digestion with restriction enzyme SfiI, which can be observed    clearly on agarose gel.

The following list of materials were used in connection with thedigestion of VHs and ligation of VHs with phagemids.

-   1. Restriction enzymes SfiI, 20000 units/ml (BioLabs, Ipswich,    Mass.) were used.-   2. T4 DNA Ligase, 400000 units/ml (BioLabs, Ipswich, Mass.) was    used.

The following list of materials were used in connection with theconcentration and desalting of ligations.

-   1. Centrifugal filter: Amicon Ultra-4 with a cutoff of 3000 MW    (Millipore, Billerica, Mass.).

The following list of materials were used in connection with cellelectroporations.

-   1. TG1 electroporation-competent cells (Stratagene, La Jolla,    Calif.).-   2. Gene Pulser/MicroPulser Cuvettes (Bio-Rad, Hercules, Calif.).-   3. Gene Pulser (Bio-Rad, Hercules, Calif.)

The following list of materials were used in connection preparing the VHlibrary.

-   1. 2YT medium: 0.5% (w/v) NaCl, 1% (w/v) yeast extract, 1.6% (w/v)    tryptone in distilled water. Autoclaved and stored at room    temperature.-   2.20% (w/v) glucose in distilled water. Sterilized using 0.22 μm    pore size filter (Nalgene, Rochester, N.Y.).-   3. M13KO7 helper phage (BioLabs, Ipswich, Mass.).-   4. Antibiotics: 100 mg/ml ampicillin and 100 mg/ml kanamycin.

A more detailed description of the specific methods that could beemployed in Example 2 are outlined as follows. Equivalent methods can beemployed as will be understood by one of ordinary skill in the art. Thisspecific listing of methods is not meant to limit the present inventionin any way.

Detailed List of Methods:

To construct a high-quality (high diversity, low mutation rate, and veryfew of reading frame shifts) antibody library, it is important tooptimize each step before next step can be performed.

Lymphocyte Isolation by Ficoll-Paque Plus Regents

-   1. To a 15 ml BD Falcon tube, add 2 ml of defibrinated or    anticoagulant-treated blood and 2 ml of balanced salt solution. Mix    by drawing the blood and buffer in and out of a Pasteur pipette.    Note that tissue culture plasticware or pretreated glassware should    be used. All glassware which comes in contact with the samples    should be siliconized before use. The glassware should be immersed    in a 1% silicone solution for 10 seconds, washed with distilled    water (6 times) and then dried in an oven.-   2. Invert the Ficoll-Paque Plus bottle several times to ensure    thorough mixing. Pipette 3 ml of the reagents into a new 15 ml BD    Falcon tube. Carefully layer the diluted blood sample (4 ml) onto    the Ficoll-Paque Plus. When layering the sample do not mix the    regents and the diluted blood sample.-   3. Centrifuge at 400 g for 30-40 minutes at 18-20° C. Note that it    is important to maintain exactly 18-20° C. temperature in the    centrifuge. Lower temperature will result in precipitates in the    layer of plasma making the lymphocyte layer unclear. Optimization of    the duration of centrifuging is also recommended to yield a clear    lymphocyte layer. This can be accomplished through a practice using    irrelevant blood samples. After centrifugation, generally four    layers can be clearly observed including plasma, lymphocyte,    Ficoll-Paque Plus, and granulocyte/erythrocyte layer from top to    bottom, respectively. Draw off the upper layer of plasma using a    clean Pasteur pipette, leaving the lymphocyte layer undisturbed at    the interface.-   4. Transfer the lymphocyte layer to a clean 15 ml BD Falcon tube    using a clean Pasteur pipette. It is critical to remove the entire    interface but with a minimum amount of Ficoll-Paque Plus and plasma.    Removing excess plasma causes contamination by platelets and plasma    proteins. Removing excess Ficoll-Paque Plus results in unnecessary    granulocyte contamination.-   5. Add at least 3 volumes of balanced salt solution to the    lymphocytes. Suspend the cells by gently drawing them in and out of    a Pasteur pipette.-   6. Centrifuge at 400 g for 10 minutes at 18-20° C. Remove the    supernatant and resuspend the lymphocytes in 6-8 ml balanced salt    solution by pipetting them gently in and out.-   7. Determine the number of living cells by using hemacytometer: Mix    50 μl cell suspension with 50 μl trypan blue stain, load 20 μl of    the mixture to the hemacytometer, count the total number of living    cells (i.e., the unstained cells, since only the live cells have    intact membrane that is not permeable for the dye), and calculate    the total cell quantity according to the hematocytometer    instructions.-   8. Centrifuge the cell suspension at 400 g for 10 minutes at    18-20° C. Remove the supernatant. The lymphocyte pellet can be used    immediately for RNA extraction or stored at −80° C. for later use.    Extraction of Total RNA from Lymphocytes

To extract the total RNA we used RNeasy Mini Kit from Qiagen followingthe basic protocol provided by the manufacturer. Note that this kitprovides enrichment for mRNA by eliminating most RNAs shorter than 200nucleotides such as 5.8S rRNA, 5S rRNA, and tRNAs. Thus, it may be moreefficient for the products to be retro-transcribed to cDNA. Below aredescribed a few modifications in this protocol that may improved theyield and quality of the extracted RNA.

-   1. Thaw the lymphocyte pellet at room temperature if it is stored at    −80° C. Note that the lymphocytes should not be frozen but used for    RNA extraction directly. Also it will be better for RNA products to    be retro-transcribed to cDNA without freeze-thaw cycle. Finish these    steps within one day. Gently tap the bottom of the tube containing    the lymphocyte pellet on the bench to loosen the cells.-   2. Disrupt cells (up to 5×10⁶) by addition of 350 μA of Buffer RLT    from RNeasy Mini Kit. Note that there are limitations with the    capacity of the buffer to lyse the cells and the binding capacity of    the column. Thus, it is important to use appropriate number of cells    in order to obtain optimal RNA yield and purity. Vortex or pipette    to mix.-   3. Pipette the lysate directly onto a QIAshredder spin column placed    in a 2 ml collection tube, and centrifuge for 2 min at 12000×rpm.-   4. Add 1 volume of 70% ethanol to the homogenized lysate and mix    well by pipetting. Do not centrifuge.-   5. Apply all sample, including any precipitate that may have formed,    to an RNeasy mini column placed in a 2 ml collection tube.    Centrifuge for 15 s at 10000×rpm. Discard the flow-through.-   6. Add 700 μA Buffer RW1 to the RNeasy column. Centrifuge for 15 s    at 10000×rpm. Discard the flow-through and collection tube.-   7. Transfer the RNeasy column into a new 2 ml collection tube.    Pipette 500 μl Buffer RPE onto the RNeasy column. Centrifuge for 15    s at 10000×rpm. Discard the flow-through.-   8. Add another 500 μl Buffer RPE to the RNeasy column. Centrifuge    for 2 min at 10000×rpm to dry the membrane in the column.-   9. Transfer the RNeasy column to a new 1.5 ml collection tube.    Pipette 30-50 μl RNase-free water onto the membrane in the column.    Centrifuge for 1 min at 10000×rpm to elute. Store the RNA product at    −80° C. and use it quickly.    Retro-transcription of RNAs to cDNAs

These instructions assume the use of SuperScript™ III First-StrandSynthesis System from Invitrogen. The following procedure is designed toconvert total RNA (5 pg to 25 μg) or mRNA (5 pg to 2.5 μg) intofirst-strand cDNA.

-   1. Mix and briefly centrifuge each component in the kit before use.-   2. Prepare two RNA/primer mixtures, one with oligo (dT)₂₀ and the    other with random hexamers, in 0.2 ml Corning® PCR tubes by    combining the following:

Total RNA x μl (up to 25 μg) 50 μM oligo (dT)₂₀ or 5 μl 50 ng/μl randomhexamers 10 mM dNTP mix 5 μl DEPC-treated water 40-x μl Total 50 μl

Incubate at 65° C. for 5 min, then place on ice for at least 1 min.

-   3. During the incubation, set up two tubes containing the same cDNA    synthesis mixtures by adding each component in the indicated order:

10 × RT buffer 10 μl 2.5 mM MgCl₂ 20 μl 0.1M DTT 10 μl RNase OUT ™ (40u/μl) 5 μl Superscript ™ III RT (200 u/μl) 5 μl Total 50 μl

-   4. Add 50 μl cDNA synthesis mixtures to the 50 μl RNA/primer    mixtures, mix gently, and collect by brief centrifugation. Incubate    as follows:    For oligo (dT)₂₀ primer: 50 min at 50° C.    For random hexamer primer: 10 min at 25° C., followed by 50 min at    50° C.-   5. Terminate the reactions at 85° C. for 5 min. Chill on ice.-   6. Collect the reactions by brief centrifugation add 5 μl of RNase H    to each reaction, and incubate for 20 min at 37° C.-   7. Run a 0.8% (w/v) agarose gel using 2 μl of the reaction to simply    check the amount and length distribution of cDNA. An example of the    results produced is shown in FIG. 21. The cDNA reactions are    combined and stored at −20° C. for further use.    PCR Amplification of CDRs and FRs    1. First Round of PCR to Get CDR2s

To amplify the CDR2s from cDNA samples, perform eight amplifications.Set up one PCR tube for each primer combination as the following:

ddH₂O 23-x μl 2 × High Fidelity PCR Master 25 μl Forward primer (25 μM)1 μl Reverse primer (25 μM) 1 μl cDNA x μl (~1 μg) Total 50 μlPrimer Recombinations:

-   -   H2-F1/H2-R1; H2-F1/H2-R2; H2-F1/H2-R5; H2-F2/H2-R3; H2-F3/H2-R1;        H2-F3/H2-R2; H2-F3/H2-R3; H2-F3/H2-R4

Perform the PCR under the following conditions:

-   -   Step 1: 4 min at 94° C. for initial denaturation    -   Step 2: 45 sec at 94° C.; 45 sec at 55° C.; 1 min at 72° C. (30        cycles)    -   Step 3: 5 min at 72° C.

Run the products separately on a 2% agarose gel to check the specificamplification of CDR2s. An example of the results produced is shown inFIG. 22A. Cut out the correct-sized bands on the gel and purify the DNAusing, for example, QIAquick Gel Extraction Kit (Qiagen, Cat. #28706).Running the eight products separately on the gel is strongly recommendedto make observation. When different templates are used, products may notbe observed on the gel for one or two primer combinations. This shouldbe due to very limited number of templates for the specific primercombinations so it is not necessary to repeat the reaction. However, itis recommended to cut out the correct-sized gel and purify the limitednumber of DNA for these primer combinations as is done to those withclear bands. This essentially helps maintain the diversity of therepertoires. Then, pool the purified DNA and quantify it by reading theoptical density (O.D.) at 260 nm (1 O.D. unit=50 μg/ml). Store thesample at −20° C. for later use.

2. First Round of PCR to Get CDR3s

Three amplifications are performed to obtain CDR3. Set up the reactionfor each primer combination and perform the PCR as it is described abovefor CDR2 amplification except for the use of different primers.

Primer Combinations:

-   H3-F1/H3R; H3-F2/H3R; H3-F3/H3R    Run the products separately on a 2% agarose gel to check the    specific amplification of CDR3s. An example of the results produced    is shown in FIG. 22B. Purify the DNA. Note that when running the    CDR3 products on 2% agarose gel, the bands will not be so sharp due    to their highly diverse lengths. Thus, cut as wide a gel as you can    to make sure it covers those CDR3s with long or short lengths. Next,    pool and quantify it by reading the optical density (O.D.) at 260 nm    (1 O.D. unit=50 μg/ml). Store the sample at −20° C. for later use.    3. First Round of PCR to Get CDR1s

Only one reaction is needed for CDR1 amplification as the following.

ddH₂O 46-x μl 2 × High Fidelity PCR Master 50 μl H1-F (25 μM) 2 μl H1-R(25 μM) 2 μl m0 x μl (~0.1 μg) Total 100 μl

Perform the PCR under the same conditions as for the CDR2 amplification.At least 1 μg of purified DNA is required in order to proceed further.

4. First Round of PCR to Get FR3

Only one reaction is needed for FR3 amplification as the following

ddH₂O 46-x μl 2 × High Fidelity PCR Master 50 μl FR3-F (25 μM) 2 μlFR3-R (25 μM) 2 μl m0 x μl (~0.1 μg) Total 100 μl

Perform the PCR under the same conditions as for the CDR2 amplification.At least 1 μg of purified DNA is required in order to proceed further.

Assembly of Entire VHs

1. Perform a Second Round of PCR (Overlap Extension) to Get CDR1S andCDR2S Together

The primers in the first round of PCR create identical sequences in thedownstream regions of the CDR1s and the upstream regions of CDR2s. Theseidentical sequences serve as the overlap for the second-round extension.

Set up a reaction without primers as the following. The CDR1s and CDR2sshould be added in the same molarities.

ddH₂O 46-x-y μl 2 × High Fidelity PCR Master 50 μl CDR1s x μl (~100 ng)CDR2s y μl (~120 ng) Total 100 μlPerform the PCR Under the Following Conditions:

-   Step 1: 4 min at 94° C. for initial denaturation-   Step 2: 45 sec at 94° C.; 45 sec at 55° C.; 1 min at 72° C. (7    cycles)-   Step 3: 5 min at 72° C.

After the cycling, add primers to the reaction: 2 μl H1-F (25 μM) and 2μl H2-R1-5 mixture (25 μM). Then perform another 15 cycles of PCR underthe same condition. Note that other than the major correct-sizedfragments, this procedure could generate minor nonspecific products forsome reasons. Optimization is may be helpful to minimize nonspecificamplification. Increasing the annealing temperature from 55° C. to 60°C. and reducing the number of cycles from 7 to 5, 15 to 12 before andafter addition of primers, respectively, may help.

2. Second Round of PCR (Overlap Extension) to Get FR3 and CDR3s Together

The procedure is almost the same as above except the use of differentgene fragments and primers for overlap. The reaction after addition ofprimers contains the following:

ddH₂O 46-x-y μl 2 × High Fidelity PCR Master 50 μl FR3 x μl (~100 ng)CDR3s y μl (~120 ng) FR3-F (25 μM) 2 μl H3-R (25 μM) 2 μl Total 100 μl

Run the products on a 2% agarose gel, purify the DNA, and quantify it.At least 2 μg each of the purified CDR1-CDR2 and FR3-CDR3DNA is requiredto proceed.

3. Third Round of PCR (Final Overlap Extension) to Get Whole-Length VHs.

Also, the procedure is almost the same as above except the use ofresultant gene fragments from Step 1 (CDR1-CDR2) and 2 (FR3-CDR3) above,and extension primers for overlap. The reaction after addition ofprimers contains the following:

ddH₂O 46-x-y μl 2 × High Fidelity PCR Master 50 μl CDR1-CDR2 x μl (~100ng) FR3-CDR3 y μl (~100 ng) H1-F (25 μM) 2 μl HISR 2 μl Total 100 μl

Run the products on a 2% agarose gel, purify the DNA with gel extractionkit (elute the DNA with ultra pure water in this step instead of elutionbuffer provided with the kit), and quantify it. At least 50 μg ofpurified VHs is needed to make a library with a size of up to 10¹⁰. Ifthe yields are too low, repeat the final overlap PCR and pool the endproducts.

Digestion of VHs and Phagemid Vector, and Ligation of Same

1. Digestion of VHs and Phagemid Vector

The reaction for VH digestion should contain:

ddH₂O 870-x μl 10 × Buffer 2 100 μl VHs x μl (up to 50 μg) SfiF (20units/μl) 20 μl BSA 10 μl Total 1000 μlSet up two reactions for phagemid digestion. Each should contain:

ddH₂O 870-x μl 10 × Buffer 2 100 μl Phagemid vectorphagemid vector x μl(up to 100 μg) SfiF (20 units/μl) 20 μl BSA (100×) 10 μl Total 1000 μl

Incubate both digests at 50° C. for 3 hours. Run the digested productson agarose gels (2% for VHs and 1% for phagemids), purify the DNA withgel extraction kit (elute the DNA with ultra pure water), and quantifyit. Note that the digestion of phagemid vectors may not be complete dueto quality of DNA. To address this problem, additional treatment may beneeded to further purify the phagemids before digestion, or use moreSfiI to digest for longer time, for example, overnight.

2. Ligation of VHs with Phagemid Vector.

Before large-scale ligation can be performed, it is recommended to testthe ligations. One test can be to assess the suitability of the vectorand inserts for high-efficiency ligation and transformation. This can beaccomplished through assembling small reactions either with vector only(test for vector self-ligation) or with both vector and insert, andtransforming chemical competent cells like DH5α. Another test can be todetermine the optimal ratio between insert and vector for the highestefficiency of ligation. This can be accomplished through assemblingsmall reactions with insert and vector in different morality ratios suchas 3:1, 2:1, and 1:1, and transforming chemical competent cells. Thehighest ligation efficiency may be obtained by using insert in two-foldmolar excess.

Assemble the Reaction as the Following:

ddH₂O 1750-x-y μl 10 × T4 ligase buffer 200 μl SfiI-digested VHs x μl(~30 μg) SfiI-digested pCom3bX y μl (~90 μg) T4 ligase (200 units/μl) 50μl Total 2000 μl

Incubate at 16° C. for 72 hours.

Concentration and Desalting of Ligated Products

Concentrate and desalt the reactions by passing through a 4 ml AmiconUltra-4 centrifugal filter with a cutoff 3000 MW:

-   1. Add all 2000 μl reactions into the filter; centrifuge at 4000×g    for 20 minutes at room temperature, Remove the flow-through to a 15    ml Falcon tube (do not discard the flow-through at this moment just    in case most of DNA is lost due to the broken membrane of the    filter).-   2. Add 3.5 ml ultra pure water into the filter and centrifuge under    the same condition for 30 min, remove the flow-through to a 15 ml    Falcon tube.-   3. Repeat Step 2 at least twice. Note that the desalting of DNA    samples is an important step to the success of electroporations.    High concentration of ions in the DNA solution will result in a long    and intense pulse in electroporations, which causes cell damage or    rupture. At least 1000-time dilution of DNA solution can be needed    to generate time constants of 4.6-5.0 s in electroporations that    generally gave the highest efficiency. In the last repeat,    centrifuge for a longer time, making sure that about 50 μl reactions    remain in the filter.-   4. Gently pipette the reactions and remove them to a 1.5 ml    Eppendorf tube, store at −20° C. for later use.    Electroporations and Preparation of Library-   1. Pre-warm 1 L 2YT medium containing 1% glucose (w/v) at 37° C.    Chill 50 gene puller cuvettes on ice. At the same time thaw, on ice,    the desalted ligations and 2 ml of TG1 electroporation-competent    cells.-   2. Divide 2 ml of TG1 competent cells into 5 pre-chilled 1.5 ml    Eppendorf tubes with 400 μl each. Add 10 μl ligations to each tube    and pipet gently to mix. Transfer 41 μl mixtures to each cuvette.    Gently tap the cuvette on the bench to make the mixture fill out the    bottom of the cuvette.-   3. Electroporate at 1.8 kV, 25 μF, and 200Ω. Flush the cuvette    immediately with 1 ml and then twice with 2 ml of pre-warmed 2YT    medium and combine the 3 ml in a 2 L flask. After all    electroporations are completed, add 850 ml pre-warmed 2YT medium    left to the flask.-   4. Shake at 250 rpm for 30 min at 37° C. Serially dilute 10 μl of    the culture in 100 μl of 2YT medium, and spread on LB agar plates    containing 2% glucose (w/v) and 100 μg/ml of ampicillin. Incubate    the plates overnight at 37° C. Calculate the total number of    transformants by counting the number of colonies, multiplying by the    culture volume, and dividing by the plating volume.-   5. Add 1 ml of 100 mg/ml ampicillin to the 1-L culture and shake for    additional 2 hours at 37° C.-   6. Take 1 ml of the culture and measure the cell density by reading    O.D.600. Calculate the total number of cells by multiplying the    O.D.600 value by 5×10⁸ (estimated number of cells in 1 ml culture    when O.D.600 reaches 1) and the culture volume (1000 in this case).    Add 10 M.O.I. of M13KO7 helper phage to the culture. Incubate at    37° C. for 30 min, shaking for homogenization every 10 min.-   7. Spin down the cells at 5000 rpm for 10 min. Resuspend in 2 L 2YT    medium containing 100 μg/ml of ampicillin and 50 μg/ml of kanamycin.    Incubate at 250 rpm overnight at 30° C.-   8. Spin at 5000 rpm for 15 min at 4° C. Save the bacterial pellet    for phagemid preparation using, for example, the Qiagen HiSpeed    Plasmid Maxi Kit. For phage precipitation, transfer the supernatant    to a clean 2 L flask and add ¼ volume of 20% (w/v) PEG8000 and 2.5 M    NaCl solution. Mix well and incubate on ice for at least 1 h.-   9. Spin at 14000 g for 20 min at 4° C. Discard the supernatant.    Resuspend the phage pellet in 50 ml PBS, pH7.4 by pipetting up and    down along the side of the centrifuge bottle by using a 10-ml    pipette.-   10. Spin at 5000 rpm for 10 min at 4° C. Transfer the supernatant to    a clean 200 ml flask and add ¼ volume of 20% (w/v) PEG8000 and 2.5 M    NaCl solution. Mix well and incubate on ice for 1 h.-   11. Spin at 14000 g for 20 min. Discard the supernatant. Resuspend    the phage pellet in 50 ml PBS, pH7.4.-   12. Spin at 5000 rpm for 10 min at 4° C. Transfer the supernatant to    a clean 200 ml flask.-   13. Add the same volume of autoclaved glycerol and mix well.-   14. Measure the concentration of phage by reading O.D.280 (1    O.D.280=2.33×10¹²/ml). Aliquot the phage to make sure that each    contains phage particles at least 100 times of the total number of    transformants (calculated in step 4). Store the phage at −80° C. The    phage library is now ready for panning.

The results of Example 2 are follows.

Results:

Design of the VH Library

To obtain a diverse VH library, an approach that grafts all three CDRsfrom several sources to the framework scaffold of m0 could have beentaken. However, the estimated diversity of such an approach could bemore than 10¹² after recombination among three CDRs, which is difficultfor artificially created phage display libraries to reach. This Exampletook the approach of mutating one of the CDRs, while grafting in theremaining CDRs from a variety of different sources. Given that the CDR1swere relatively more conserved in both their sequences and theirlengths, this Example focused on grafting in CDR2s and CDR3s from avariety of sources, while randomly mutating four putativesolvent-accessible positions of the CDR1 of m0 to A/D/S/Y (thesolvent-accessible positions being amino acid residue #27, 29, 31 and 32under the IMGT numbering system) (see FIG. 5) in the original CDR1 of m0to A/D/S/Y .? not sure whether you meant our 2008 PNAS paper or thisone—you could retain it but the way it is cited appears like ours.

In order to access as many different human VH gene segments as possible,a new set of primers (see Table 1) was designed for amplification ofCDR2 and CDR3 repertoires, respectively, based on human VH germlinesequences loaded in the IMGT database(http://imgt.cines.fr/textes/IMGTrepertoire/Proteins/alleles/human/HuAl_(—)list.html).The target sequences (see italicized nucleotide sequences flanking CDR2and CDR3 regions in the m0 template of FIG. 5) for these primers aremostly conserved within each group of germlines. Thus, these primers, incombination with each other (see Table 2), should allow efficientamplification of all commonly used human VH gene segments.

To make the resultant library more suited for selection of antibodiesagainst a wide range of antigens including, pathogen and cancer-relatedantigens, an IgM-derived library from cord blood was used as a templateadditionally, which we assumed should provide more naïve CDRrepertoires. Given the special case of HIV-1, which has mostly evolvedto escape the human immune system, an immune library from HIV-1 patientswas also used.

Construction of the VH Library

The VH library was constructed in three steps. In the first step, 8 and3 PCRs were performed for amplification of CDR2 and CDR3 gene segmentsfrom each library template, respectively (see FIG. 6A). Products ofCDR2s from different libraries were pooled. Products of CDR3amplifications were also pooled. CDR1 repertoire was amplified from m0master VH using a degenerate primer covering the whole CDR1. FR3 segmentwas also obtained from m0 for assembly of entire VHs. In the secondstep, overlapping PCRs were performed to join CDR1s to CDR2s and FR3 toCDR3s, respectively (see FIG. 6B). In the third step, entire VHs wereassembled from the products of the second step by overlapping PCR (seeFIG. 6C). The products were cloned into phagemid vectors and a libraryof around 2.5×10¹⁰ members was obtained by performing 100electroporations as described above.

Sequence Diversity of the Library

To assess the sequence diversity of the library, 190 clones wererandomly selected from the library and sequenced. 166 complete sequenceswere obtained, of which 143 have correct reading frames. The 143sequences were aligned, and the occurrence of A/D/S/Y in each mutatedposition within CDR1s, the origins of CDR2s and their mutations, and thelength distribution of CDR3s were tabulated. The results showed thatthese sequences were totally different from each other afterrecombination among three CDRs although several identical CDR1s andCDR2s were found due to the limited diversity or conservationthemselves.

For CDR1s, A/D/S/Y was well scattered in each position while Y dominatedslightly (see FIG. 8A). CDR2s were derived from VH1, 2, 3, 4, 5 and 7 ofgermlines (see FIG. 8B). VH4-derived CDR2s were dominant by coveringaround 50% of the 143 sequences. No CDR2s originated from VH6, which isthe smallest one, were found. More than 40% of CDR2s are completelyidentical to the germline sequences, while there are members with manymutations, regardless of those induced by PCR process (see FIG. 8C). Forthe CDR3s, the lengths vary from 5 to 24 amino acid residues with acentral peak from 12 to 14 residues and a side peak at 17 residues (seeFIG. 8D). In comparison with the length distribution of CDR3s in vivo(Wu et al., Proteins, 1993), there is relatively limited number of CDR3swith length shorter than 8 or longer than 19 residues. Moreover, CDR2sfrom different origins were paired with CDR3s with varying length (seeFIG. 9). These data suggest that the library should have high sequencediversity.

Folding of Phage-displayed VHs

The folding of phage-displayed VHs was evaluated by measuring theiractivity binding to protein A since a VH3 framework scaffold m0 was usedin library construction. The library was cycled through 4 rounds ofselection to enrich for members that were capable of binding to proteinA, and thus, were likely to be folded as natural VH3 structures. 95clones were randomly picked from the fourth round of enrichment andsequenced and 79 complete and clear sequences were obtained. In the 143clones from the original library, all 4 positions mutated in CDR1s biasslightly toward residue Y (see FIG. 8A) and VH3-originated CDR2s coverabout 20% (see FIG. 8B). In contrast, all 79 sequences picked after thebinding selection process against protein A were shown to have CDR2sderived from VH3. Within CDR1, residue D apparently dominates inposition #27, 29 and 32 (see FIG. 10). CDR3 of these clones are muchdiversified and the length varies from 7 to 17 residues.

VH Expression and Solubility

We evaluated the expression and solubility of VHs by measuring the yieldof soluble proteins purified from the soluble fraction of E. coliperiplasm. First, 2 clones each, one with relatively short CDR3 and theother long CDR3, were selected from those with CDR2s derived from 6different groups of human VH germlines, respectively, expressed andpurified as described above. The SDS-PAGE result showed that 11 of 12could be expressed in the form of soluble proteins and the estimatedyield ranged from 0.5 to 24 mg 1⁻¹ (see FIG. 11A). There was nosignificant relation between the yield and the CDR2 origins or CDR3lengths of VHs tested.

Then, 12 clones (named c3, c6, d1, d7, b4, c11, d10, b3, b5 (alsoreferred to hereas m36), b7, g6 and e11) with CDR3 length from 7 to 24residues were picked from those with VH4-derived CDR2s and measured forsoluble protein expression. 10 of 12 clones gave yield varying from 0.5to 15 mg 1⁻¹ and there was no obvious connection between the yield andthe lengths of CDR3 either (see FIG. 11B). Last, we evaluated thesolubility of VHs selected after three and four rounds of biopanning ofthe library with HIV-1 antigen gp120-CD4 fusion protein and gp140(described further below).

Twelve positive clones on monoclonal phage ELISA with A405 of >1.5 wereexpressed and purified. As shown in FIG. 11C (which is a duplicate ofthe gel image of FIG. 7 showing the identify of clone b5 or “m36”), 11of 12 clones gave high-level yield from 5.0 to 30 mg 1⁻¹. These cloneshave CDR2s that originated from VH3, VH4 and VH7, which were determinedto be dominant in the library (see FIG. 8B). These clones were alsoshown to have medium length CDR3s.

These results indicate that the framework scaffold used for libraryconstruction could be flexible enough to harbor CDR2s derived from avariety of human VH germlines and CDR3s with length as short as 7residues and as long as 24 residues.

Selection of VHs Against Viral and Cancer-related Antigens

To assay the performance of the library and given the constant need fornew and better therapeutics, the library was panned against three viralantigens and one cancer-related antigen (see Table 3).

TABLE 3 Panning of the library with viral and cancer-related antigens.Positive No. of unique clones/ clones/ EC₅₀ of the No. of testedsequenced best binder Antigen selections clones clones (μg/ml) gp120-CD44 188/190  14/14 ND (HIV-1 antigen) gp120-CD4/ 4 17/190 7/7 0.040^(a)gp140 (HIV-1 antigen) B5R (vaccinia 3 6/95 6/6 0.013 antigen) Her2(cancer 3 5/95 5/5 1.1 antigen) ^(a)Binding activity with gp120-CD4.

Selections were carried out using antigens conjugated on magnetic beadsas described above. In the sequential panning with HIV-1 antigens(alternate panning between HIV gp120-CD4 and gp140), gp120-CD4 was usedfor the first round and third round, and gp140 the second round andfourth round. Enrichment was achieved in all selections and positiveclones were identified on monoclonal phage ELISA.

A handful of clones having a high value of A405 following the ELISA weresequenced. All of the sequenced clones were mosaic, especially in CDR1and CDR3 (see Table 4).

TABLE 4 CDR diversity of phage-displayed VHs selected from biopanning.Round CDR2 Antigen No. Clone CDR1 Sequence Origin CDR3 gp120- 3 a9D-H-HS-- INHSGIT  4-4, 4-34 AIVDTAMVWDY CD4 (SEQ ID NO: 23)(SEQ ID NO: 44) b2 S-A-SD-- INHSGST  4-4, 4-34 AASGSYSDY (SEQ ID NO: 24) (SEQ ID NO: 45) b3 A-D-SY-- INHSGST  4-4, 4-34ATHDYGDSFES (SEQ ID NO: 24) (SEQ ID NO: 46) c3 D-S-YS-- INHSGST 4-4, 4-34 ARIGDGFFSDAFEI (SEQ ID NO: 24) (SEQ ID NO: 47) c6 D-Y-DY--IDNSGST  4-28, 4-30-2, ect. AGDYGSGSEFEN (SEQ ID NO: 25) (SEQ ID NO: 48)d1 D-D-YD-- INTDGDIP 7-4-1 AKYTWNSDSGWGEL (SEQ ID NO: 26)(SEQ ID NO: 49) d7 Y-D-YD-- IYHRGNT 4-4, 4-30-2, ect.  VGYGADQDDC(SEQ ID NO: 27) (SEQ ID NO: 50) e10  D-A-DD-- INHTGST 4-34 ATHDYGDSFES(SEQ ID NO: 28) (SEQ ID NO: 51) g2 D-D-DY-- INHSGST   4-4, 4-34AADTGNAFDI (SEQ ID NO: 24) (SEQ ID NO: 52) 4 a11 Y-D-DD-- INHSGST 4-4, 4-34 AGSSGWLHEY (SEQ ID NO: 24) (SEQ ID NO: 53) b4 D-D-AS--INHSGST   4-4, 4-34 ATDQAGIEH  (SEQ ID NO: 24) (SEQ ID NO: 54) c8S-S-AD-- INHSGST   4-4, 4-34 ATSVGYEEL  (SEQ ID NO: 24) (SEQ ID NO: 55)c11 A-S-DY-- INHSGST   4-4, 4-34 AMSDGYSATDV (SEQ ID NO: 24)(SEQ ID NO: 56) d10 A-A-DD-- ITGSGDTT 3-23 ALTDSSSYDY (SEQ ID NO: 29)(SEQ ID NO: 57) gp120- 3 a8 D-D-SD-- INHSGST   4-4, 4-34 AFYMRGAILEYCD4/ (SEQ ID NO: 24) (SEQ ID NO: 58) gp140 b3 Y-D-SS-- INHSGST 4-4, 4-34AAYDFWSGSYPEY (SEQ ID NO: 24) (SEQ ID NO: 59) b5 A-D-SD-- INDSGNT4-34, 4-55 AIYGGNSGGEY (SEQ ID NO: 30) (SEQ ID NO: 60) b7 D-S-DY--VGGSGERT 3-23 ARIDRDGDEH (SEQ ID NO: 31) (SEQ ID NO: 61) c6 S-D-DY--INHSGST   4-4, 4-34 AGSGSYSDY  (SEQ ID NO: 24) (SEQ ID NO: 62) 4 g6D-A-DY-- INSNGSVT 3-74 ARDWGYSPED (SEQ ID NO: 32) (SEQ ID NO: 63) e11Y-D-YD-- ISYDGSIK 3-30, 3-30-3 N/A (SEQ ID NO: 33) B5R 3 z1 S-Y-DY--IYHSGST  4-30-2 ARTPPRIAAAGMR (SEQ ID NO: 34) YFDL  (SEQ ID NO: 64) z2S-D-AD-- INSSSSYI 3-21 ARDWGYSPED (SEQ ID NO: 35) (SEQ ID NO: 65) z3D-D-YS-- ISGDGGAT 3-23 ARADYRSTDH (SEQ ID NO: 36) (SEQ ID NO: 66) z4D-S-YD-- IYYSGST   4-39 ARQVAAPV  (SEQ ID NO: 37) (SEQ ID NO: 67) z5D-S-YY-- IKQDGSVV 3-7 ARDWGYSPED (SEQ ID NO: 38) (SEQ ID NO: 68) z6Y-D-YA-- ISYDGSNK 3-30, 3-30-3 VRDWGYNPED (SEQ ID NO: 39)(SEQ ID NO: 69) Her2 3 z7 Y-D-SS-- IYSGGTT   3-53, 3-66 VRDWGYNPED(SEQ ID NO: 40) (SEQ ID NO: 70) z8 Y-Y-DY-- ISNSGGTI 3-11 ARGTGLHDYGDY(SEQ ID NO: 41) WAHTEFDY  (SEQ ID NO: 71) z9 D-A-AD-- IYSGGST 3-53, 3-66 ARDWGYSPED  (SEQID NO: 42) (SEQ ID NO: 72) z10 D-D-YD--IRYDGSNK 3-30, 3-30-3, ect. ARGVDYGDYGGY (SEQ ID NO: 43) FDY (SEQ ID NO: 73)

For the panning against HIV-1 antigens, however, most of selected cloneshave CDR2s originated from VH4, in particular VH4-4 or VH4-34 sub-group.Their CDR3s vary and lengths range from 9 to 14 residues. In contrast,most and all clones selected from B5R and Her2 panning, respectively,have VH3-derived CDR2s. Some of them, for example, z2, z5 and z6, havesimilar CDR3s.

Some clones were expressed and purified, and their binding andspecificity were confirmed by ELISA against the antigen used for itsselection, as well as several unrelated antigens (e.g. BSA or antigenswhich are significantly heterologous to the one used in the panningcould be used as unrelated antigens. For example, in the Her2 panningHIV-1 antigen Bal gp120-CD4 could used as a negative control while inthe Bal gp120-CD4 panning Her2 could be used as an unrelated antigen).

In the sequential panning, an antibody with affinity in nanomolar range(EC₅₀=0.040 μg/ml), designated b5 (see FIG. 11C) (later designated m36)was identified as binding to HIV-1 antigen gp120-CD4 but neither gp120nor CD4 alone.

Further characterization showed that b5 (m36) was a potent neutralizingantibody which targeted an epitope on HIV-1 gp120 whose accessibilityappeared to be induced upon CD4 binding to HIV-1 gp120. In addition, b5(m36) was determined to be cross-reactive against HIV-1 isolates fromclade A, B, C, and D (see FIG. 17).

Regarding panning results for vaccinia antigen B5R, a specific VH, z1,was found with an EC₅₀ of 0.013 μg/ml. Binders against humanself-antigen Her2 could also be selected, albeit at a somewhat lowerfrequency and with lower affinity (see Table 3).

VH Oligomerization

Size exclusion chromatography was performed to measure theoligomerization of VHs using superdex75 column. 5 purified VHs wererandomly selected from 12 positive clones (c3, c6, d1, d7, b4, c11, d10,b3, b5, b7, g6 and e11) against HIV-1 antigen gp120 on monoclonal phageELISA and tested for oligomerization. 2 of 5 VHs, e.g. b7 and d10, haveCDR2s originated from human VH3 while the CDR2s of the other 3 fromhuman VH4 (see FIG. 11C).

Before conducting the experiments, all 12 VHs (c3, c6, d1, d7, b4, c11,d10, b3, b5, b7, g6 and e11) had already been stored at 4° C. for 4weeks and no precipitation was observed with these protein solutions.The data shown in FIG. 12 demonstrates the existence of a monomericstate of all 5 VHs (˜15 kDa) and lack of higher order oligomers.

The results of Example 2 are further discussed as follows.

Discussion:

Domain antibodies (dAbs) are of great interest for biomedicalapplication due to their special biophysical properties. However, thepoor solubility and stability of most dAbs, particularly those derivedfrom human antibodies, severely limits the successful development ofdAbs and dAb libraries. Camels produce functional antibodies devoid oflight chains, V_(H)Hs, which are adapted replacing aliphatic residues inthe former light chain interface by hydrophobic residues, packingagainst the framework and stabilizing the global V_(H)H fold by longCDR3s (Holt et al., Trends Biotech., 2003; Muyldermans et al., Trends inBiochem. Sciences, 2001). It has been demonstrated that the variabledomains of human VH3 are significantly more soluble and stable thanthose from any other human VH and VL families in the absence of lightchains (Holt et al., Trends Biotech., 2003).

Jirholt et al. used a camelized human VH3 germline, DP47, as a masterframework for construction of a dAb library of 9×10⁶ members but nofurther characterization was described in terms of solubility andfunctionality of dAbs from the library (Jirholt et al., Gene, 1998).

The present inventors have constructed a large non-immune human Fablibrary (˜1.5×10¹⁰ members) from the lymph nodes, spleen and peripheralblood lymphocytes of 50 donors. One of these Fabs (R3H1, see FIG. 4) hada stop codon in the light chain but was still isolated from librarypanning and was functional as a heavy chain alone. The VH domain of thisantibody was cloned to form the VH domain antibody, m0 (as shown in FIG.5). It was found that m0 exhibits high levels of expression and highsolubility.

m0, a natural VH domain and belonging to the VH3 family, was used as aframework to construct a large human VH domain library (˜2.5×10¹⁰members) by grafting in vivo-formed CDR2s and CDR3s from our other humanantibody libraries and mutating four residues in CDR1. The libraryexhibited a high degree of variability and most of VHs were highlysoluble. The quality of the library was also validated by selection ofVHs against a panel of antigens, including HIV-1 antigens (Balgp120-CD4; Bal gp120-CD4/R2 gp140), a vaccinia antigen (B5R), and acancer antigen (Her2).

Without wishing to be bound by theory, naturally occurring and proofreadCDRs are advantageous as compared to synthetic repertoire CDRs becausethey are processed and adapted by the immune system with respect totheir functionality and immunogenicity. Abergel and Clayerie (Eur. J.Immunol., 1991) showed that VH genes rearranged in vivo have CDR3slargely derived from the D-segments encoding amino acids with apropensity for loop formation. By comparison with synthetic CDR3s,therefore, naturally occurring CDRs may be more likely to fold properlyor produce usefully shaped binding sites. Furthermore, there is evidencethat antibodies derived from recombination of natural CDRs exhibit lowimmunogenicity (Soderlind et al., Nature Biotech., 2000). A scFv librarycontaining 2×10⁹ members has recently been constructed by randomlyshuffling all six in-vivo formed CDRs into a single master frameworkusing human VH DP47 and VL DPL3. Ten scFvs selected from the libraryspecific for different antigens did not show an increased presence ofT-cell epitopes, and they contained even fewer T-cell epitopes than thenatural antibody variable regions.

The size and sequence diversity are thought to be key features ofhigh-quality libraries. A finding consistent with theoreticalconsiderations is that the affinity of antibodies selected isproportional to the size of the library, with K_(d)s ranging from 10⁶⁻⁷for the smaller libraries to 10⁹ for the larger ones, and antibodieswith affinity comparable to those obtained from immune libraries can beselected from naïve libraries that are large enough (Andrew et al., J.Immunol. Methods, 2004).

Aiming in part at generating a library with enormous diversity, thepresent invention relates to the design of a new set of primers to allowamplification of as many VH genes as possible. The complexity of thelibrary formed according to the invention was determined by sequencing190 clones randomly selected (see FIG. 8 and FIG. 9). The 143 completesequences have CDR2s derived from 6 (VH1-5 and 7) of 7 human VHgermlines (see FIG. 8B). Since VH6 is the smallest group of germlines,we assume that CDR2s with VH6 origin could also be found if more clonesare sequenced. The length, sequence, and pairing of CDR3s with differentgermline-derived CDR2s were varied simultaneously (see FIGS. 8D and 9).These data suggest that this library should have high sequencediversity.

In a scFv library previously constructed, primers were designed foramplification of naturally occurring CDRs, but they were specific forDP47 and DPL3 frameworks only (Soderlind et al., Nature Biotech., 2000;Jirholt et al., Gene, 1998). By contrast, the primers used in thepresent invention advantageously may cover CDRs derived from all groupsof human VH germlines, as described above. They may, therefore, thepresent invention, in part, facilitates more rapid and efficientconstruction of large human VH domain libraries.

While the combinatorial strategy provides a powerful tool for creatingtremendous diversity, the m0 framework scaffold used in the libraryconstruction plays a challenging role because it must support the CDRsfrom various non-VH3 germlines while maintain a stable tertiary fold.Structural incompatibility between these foreign CDRs and the fixedframework could potentially prevent the formation of stable and solubleVHs. Thus, it is surprising and unexpected that the domain antibodies ofthe invention specific against various antigens and having a diversityof non-VH3 CDRs possess good folding characteristics.

Protein A binding is considered a marker for proper folding of human VH3(Potter et al., J. Immunol., 1996). To see whether recombinant VHs witha fixed m0 which is close to VH3 scaffold could retain activity, thelibrary was cycled through four rounds of selection against protein A.All 79 clones randomly picked from the fourth round of selection hadCDR2s derived from VH3, suggesting that VH3-derived CDR2s and theirflanking FR regions are essential for their property of protein Abinding, in agreement with previous studies (Potter et al., J. Immunol.,1996; Randen et al., Eur. J. Immunol., 1993). Interestingly, position#27 and 29 in CDR1s significantly biases toward residue D in thoseclones after protein A selection while residue Y dominates in thesepositions of VHs with VH3-derived CDR2s before selection (see FIG. 10).There is also a slight increase in the usage of D in position #32 andthis does not happen to position #31. Given D is a small and polarresidue, and Y an aromatic residue, the polarity of the residues inthese positions could significantly impact the folding of VH3 withrespect to protein A binding activity.

It has recently been demonstrated that this property can be used tomonitor the structural stability and soluble expression of VH3 (Bond etal., J. Mol. Biol., 2003). Thus, the fact that VHs with non-VH3 CDR2slack the property further raises a doubt whether these VHs can besoluble and stable. To address this issue, the yield of soluble VHshaving non-VH3 CDR2s and CDR3s ranging from 7 to 24 residues wasmeasured (FIG. 11). The results show that most of VHs have favorableyield of soluble proteins and the yield is not significantly related toCDR2 origin and CDR3 length.

In addition to proper folding, the domain antibodies of the inventionwere evaluated as to whether they possessed any tendency to form dimersand higher order multimers—which, for steric considerations, may not bedesirable. Antibodies in the format of scFv have the tendency to formdimers and higher order multimers in a clone-dependent and relativelyunpredictable way (Abergel et al. 1991; Andrew et al., 2004; Potter etal., 1996). The oligomerization of 5 VHs randomly selected was measuredby size exclusion chromatography and they all appeared to be monomeric(see FIG. 12). There is evidence that VHs are prone to aggregate uponconcentration or prolonged standing at 4° C. (Kortt et all, 1995; Ewertet al., 2002). The 5 VHs tested for oligomerization were concentrated toget concentrations of as high as 10 mg/ml. After being stored at 4° C.for more than 8 weeks no precipitation was observed with these 5 proteinsolutions.

These data suggest that the m0 scaffold not only is capable ofpresenting diverse CDRs, but also has desirable properties forbiotechnological application, including, high level of expression,solubility, resistance to aggregation in solution, and lack ofpolymerization.

A central question in the evaluation of the library is whetherhigh-affinity functional VHs could be directly selected against a panelof antigens. The library design was based on the principle that aspecific single framework had the potential to include CDRs derived fromother groups of germline genes and the small antigen-binding surface wascapable of presenting structural diversity enough to form paratopes fora wide range of antigens.

Biopanning of the library showed that VHs with estimated affinity innanomolar range could be obtained against viral antigens (see Table 3).Binders against human self-antigens could also be selected, albeit at asomewhat lower frequency and with lower affinity. These binders are muchdiversified in CDRs (see Table 4) suggesting that a number of differentantibody specificities could be generated. Thus, this Exampledemonstrates that this library could be useful for selection ofhigh-affinity binders as therapeutics, e.g., HIV therapeutics.

Example 3 Human Domain Antibodies to Conserved Sterically RestrictedRegions on gp120 as Exceptionally Potent Cross-reactive HIV-1Neutralizers

This Examples describes the identification and characterization of anantibody heavy chain variable domain (VH) (domain antibody, dAb), m36,targeting highly conserved but sterically restricted CD4-induced (CD4i)structures on the Env. It is believed that M36 is the first reportedrepresentative of a novel class of potent and broadly cross-reactiveHIV-1 inhibitors based on human dAbs. It has potential as a candidatetherapeutic and a microbicide, and as an agent for exploration of thehighly protected conserved Env structures with implications for thedesign of novel small molecule inhibitors, and elucidation of themechanisms of virus entry into cells and evasion of immune responses.

The following methods and materials were employed in this Example. Theresults of this Example are subsequently discussed below.

Methods and Materials:

Cells, Viruses, Plasmids, gp120, gp140 and Antibodies

293T cells were purchased from ATCC. Other cell lines and plasmids usedfor expression of various HIV-1 Envs were obtained from the NIH AIDSResearch and Reference Reagent Program (ARRRP). Recombinant gp140s werekindly provided by C. Broder (USUHS, Bethesda, Md.). Gp120_(Bal) and thesingle-chain fusion protein gp120_(Bal)-CD4 (8) were gifts from T. Fouts(Institute of Human Virology, Baltimore; currently at Profectus,Baltimore, Md.). Horseradish peroxidase (HRP)-conjugated anti-FLAG tagantibody and HRP-conjugated anti-human IgG (Fc-specific) antibody werepurchased (Sigma-Aldrich, St. Louis, Mo.).

Library Construction and Selection of VHs Against Hiv-1 Antigens

A large phage-displayed human VH library (˜2.5×10¹⁰ individuals) wasconstructed by grafting of naturally occurring heavy chain CDR2s andCDR3s from other five template libraries to a VH framework scaffold, m0,and randomly mutating four putative solvent-accessible residues in itsCDR1 (FIG. 23). These template libraries include: (a) a naive human Fablibrary (5×10⁹ members) from peripheral blood B cells of 10 healthydonors (Zhu et al., 2006); (b) a naïve human Fab library (1.5×10¹⁰members) from peripheral blood B cells of 22 healthy donors, spleens ofthree donors, and lymph nodes of healthy 34 donors; (c) two naïve humanFab libraries (6×10⁸ and 7.2×10⁸ members, respectively) from cord bloodof two healthy babies, respectively; and (d) an immune human Fab libraryfrom the bone marrow of three long-term nonprogressors whose seraexhibited the broadest and most potent HIV-1 neutralization among 37HIV-infected individuals (Chen et al., J. Mol. Biol., (2008)382:779-789, which is incorporated herein by reference in its entirety).This dAb library was used for selection of VHs against HIV-1 antigensconjugated to magnetic beads (Dynabeads M-270 epoxy; DYNAL Inc., NewHyde Park, N.Y.) as described previously (Zhu et al, 2006). Forsequential panning, 10 and 5 μg of gp120_(Bal)-CD4 was used in the firstround and third round, respectively; antigens were alternated with 5 μgof gp140_(R2) or gp140_(JRFL) during the second round and fourth round.Clones that bound to HIV-1 antigens were identified from the third andfourth round of biopanning by using monoclonal phage ELISA as described(Zhu et al, 2006).

Construction and Cloning of m36 Fusion Proteins

The following primers were used:

m36F, (SEQ ID NO: 74) 5′-TGGTTTCGCTACCGTGGCCCAGCCGGCCCAGGTGCAGCTGGTG-3′(sense); m36F1, (SEQ ID NO: 75)5′-TGGTTTCGCTACCGTGGCCCAGGCGGCCCAGGTGCAGCTGGTG-3′ (sense); m36R1,(SEQ ID NO: 76) 5′-GTGAGTTTTGTCGGGCCCTGAGGAGACGGTGAC-3′ (antisense);m36R2, (SEQ ID NO: 77) 5′-TGGTTGTGGTTGGGGTATCTTGGGTTCTGAGGAGACGGTGAC-3′(antisense); m36R3, (SEQ ID NO: 78)5′-GTCACCAAGTGGGGTTTTGAGCTCTGAGGAGACGGTGAC-3′ (antisense); m36R4,(SEQ ID NO: 79) 5′-TTCTCGGGGCTGCCCTGAGGAGACGGTGAC-3′ (antisense); m36R5,(SEQ ID NO: 80) 5′-CAGGAGTTCAGGTGCTGAGGAGACGGTGAC-3′ (antisense); CH3F,(SEQ ID NO: 81) 5′-GGGCAGCCCCGAGAACCA-3′ (sense); CH3R, (SEQ ID NO: 82)5′-GTGGTGGTGGTGGTGGCCGGCCTGGCCTTTACCCGGAGACAG-3′ (antisense); FcF1,(SEQ ID NO: 83) 5′-ACGTGTCCCAAATGTCCAGCACCTGAACTCCTGGGG-3′ (sense);FcF2, (SEQ ID NO: 84) 5′-CCGTGCCCACGGTGCCCAGCACCTGAACTCCTGGGG-3′(sense); FcF3, (SEQ ID NO: 85) 5′-GCACCTGAACTCCTGGGG-3′ (sense); FcR,(SEQ ID NO: 86) 5′-GTCGAGGCTGATCAGCGG-3′ (antisense); FcR1,(SEQ ID NO: 87) 5′-CTCCTATGCGGCCGCTTTACCCGGAGACAG-3′ (antisense); HcF,(SEQ ID NO: 88) 5′-CCCCAACCACAACCAAAACCACAACCACAACCACAACCACAACCAAAACCAC-3′ (sense); HcR, (SEQ ID NO: 89)5′-TGGACATTTGGGACACGTGCATTCTGGTTCAGGTTTTGGTTGTGGTT TTGGTTGTGG-3′(antisense); HhF, (SEQ ID NO: 90)5′-ACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCC CAAA-3′ (sense); HhR,(SEQ ID NO: 91) 5′-TGGGCACCGTGGGCACGGGGGAGGTGTGTCACAAGATTTGGGCTCTGGGCA-3′ (antisense); HSAPR2, (SEQ ID NO: 92)5′-CTCCTATGCGGCCGCATCATCGTCGCCCCACAAACACCCCCAGCGT GGCAA-3′ (antisense);HSAPR3, (SEQ ID NO: 93)5′-CCCCCAGCGTGGCAAACATATATCTTCTGGGTGGCGCTGGCCCTTA TCGTCATC-3′(antisense).

For construction of m36CH3, the m36 gene was amplified by PCR (primer:m36F1 and m36R4) with m36-encoding plasmid pCom36 as a template. The CH3gene of human IgG1 was PCR (primer: CH3F and CH3R) amplified fromplasmid pSecTagB-Fc, which encoded the human IgG1 Fc portion (FIG. 29).M36 fragment was joined to CH3 by overlapping PCR performed in a volumeof 100 μl using both templates (in the same molarities) for 7 cycles inthe absence of primers and additional 15 cycles in the presence ofprimers (500 pM of m36F1 and CH3R, respectively). The m36CH3 productsappended with SfiI restriction sites on both sides were digested andcloned into a phagemid vector (FIG. 22). To generate m36b0Fc, m36 genewas amplified by PCR using primer m36F1 and m36R5. Human IgG1 Fc genewas obtained by PCR amplification using pSecTagB-Fc as the template andprimer FcF3 and FcR1. M36 fragment was joined to Fc as described above.The products were digested with SfiI and NotI, and cloned into pZYD-N1,which was developed by the inventors to have a NotI site after the FLAGtag. The vector pSecTagB-Fc was used for construction of m36h1Fc,m36c2Fc, and m36h3Fc. The m36 fragment was PCR amplified using primerm36F and m36R1, digested with SfiI and ApaI, and cloned into pSecTagB-Fcto generate m36h1Fc. M36c2Fc was cloned by amplifying m36 fragment(primer: m36F and m36R2), human IgG1 Fc (primer: FcF1 and FcR), andcamel IgG2 hinge (primer: HcF and HcR). The m36 fragment was fused tocamel IgG2 hinge and the product was subsequently joined to Fc byoverlapping PCR. The resultant full-length m36c2Fc product was digestedwith SfiI and PmeI, and cloned into pSecTagB-Fc vector. In the same waym36h3Fc was constructed except for the use of primer m36F and m36R3 form36 amplification, primer FcF2 and FcR for human IgG1 Fc amplification,and primer HhF and HhR for human IgG3 hinge amplification. To generatem36SAbp, the m36 fragment was amplified using primer m36F1 and HSAPR3,purified and further extended by PCR using primer m36F1 and HSAPR2. Theproducts were digested with SfiI and NotI, and cloned into pZYD-N1.

Expression and Purification of m36 and its Fusion Proteins

M36, m36SAbp, m36CH3 and m36b0Fc were expressed in E. coli HB2151 asdescribed previously (Zhu et al, 2006). The bacterial pellet wascollected after centrifugation at 5,000×g for 10 min and resuspended inPBS (pH 7.4) containing 0.5 mU polymixin B (Sigma-Aldrich, St. Louis,Mo.). After 30 min incubation with rotation at 50 rpm at roomtemperature, it was centrifuged at 25,000×g for 25 min at 4° C. Thesupernatant was used for purification of m36, m36SAbp and m36CH3 byimmobilized metal ion affinity chromatography (IMAC) using Ni-NTA resin(Qiagen, Valencia, Calif.) according to manufacturer's protocols. Forpurification of m36b0Fc, nProtein A Sepharose 4 Fast Flow (GEHealthcare, Piscataway, N.J.) was used. M36h1Fc, m36c2Fc and m36h3Fcwere expressed in 293 free style cells. CellFectin (Invitrogen,Carlsbad, Calif.) was used to transfect 293 free style cells accordingto the instructions of the manufacturer. Three days posttransfection,the culture supernatant was harvested and used for purification ofm36h1Fc, m36c2Fc and m36h3Fc by using nProtein A Sepharose 4 Fast Flow.

Binding ELISA

Antigens were coated on Corning EIA/RIA high-binding 96-well plates(Corning Inc., Corning, N.Y.) at 50 ng per well overnight at 4° C. andblocked with 3% nonfat milk in PBS. Threefold serially diluted antibodywas added in the absence or presence of sCD4 at 2 μg/ml finalconcentration and incubated at room temperature for 2 h. The plates werewashed with PBS containing 0.05% Tween 20. Bound m36, m36SAbp or m36CH3was detected by HRP-conjugated anti-FLAG tag antibody (Sigma-Aldrich,St. Louis, Mo.). The m36 fusion proteins with human IgG1 Fc weredetected by HRP-conjugated anti-human IgG (Fc-specific) antibody(Sigma-Aldrich, St. Louis, Mo.). The assay was developed at 37° C. withABST substrate (Roche, Indianapolis, Ind.) and monitored at 405 nm. Thehalf-maximal binding (EC₅₀) was calculated by fitting the data to theLangmuir adsorption isotherm.

Competition ELISA

Antigens were coated and blocked as described above. M36 at aconcentration leading to 90% maximum binding was premixed with threefoldserially diluted competitors without or with sCD4 at 2 μg/ml finalconcentration. Mixtures were subsequently added to each well andincubated. Bound m36 was detected and the assay was developed asdescribed above.

Measurement of m36 Oligomerization

Superdex75 column was calibrated with protein molecular mass standard of14 (ribonuclease A), 25 (chymotrypsin), 44 (ovalbumin), 67 (albumin),158 (aldolase), 232 (catalase), 440 (ferritin) and 669 (thyroglobulin)kDa. Purified m36 in PBS were loaded onto the column that had beenpre-equilibrated. The proteins were eluted with PBS at 0.5 ml/min.

Pseudovirus Neutralization Assay

Viruses pseudotyped with HIV-1 Envs were prepared by cotransfection of70-80% confluent 293T cells with pNL4-3.luc.E-R- and pSV7d constructsencoding HIV-1 Envs using the PolyFect transfection reagent (Qiagen,Valencia, Calif.) according to manufacturer's instruction. Pseudotypedviruses were obtained after 24 h by centrifugation and filtration ofcell culture through 0.45-μm filters. For neutralization, viruses weremixed with different concentrations of antibodies and/or sCD4 at 8 nMfor 1 h at 37° C., and then the mixture was added to ˜1.5×10⁴HOS-CD4-CCR5 (used for all R5 and dual tropic viruses) or HOS-CD4-CXCR4cells grown in each well of 96-well plates. Luminesence was measuredafter 48 h using the Bright-Glo Luciferase Assay System (Promega,Madison, Wis.) and a LumiCount microplate luminometer (Turner Designs).Mean relative light units (RLU) for duplicate wells were determined.Percentage inhibition was calculated by the following formula:(1−average RLU of antibody-containing wells/average RLU of virus-onlywells)×100. IC₅₀ and IC₉₀ of neutralization were assigned for theantibody concentration at which 50% and 90% neutralization wereobserved, respectively.

The results of Example 3 are provided as follows.

Results:

Selection of m36 from a Newly Constructed Human Antibody VH Library.

Examples 1 and 2 discuss the identification of a phage-displayed heavychain only antibody by panning of a large (size ˜1.5×10¹⁰) human naiveIgM library against an Env. The VH of this Fab, designated as m0, wasindependently folded, stable, highly soluble, monomeric, and expressedat high levels in bacteria. M0 was used as a scaffold to construct alarge (size ˜2.5×10¹⁰) highly-diversified phage-displayed human VHlibrary by grafting naturally occurring CDR2s and CDR3s of heavy chainsfrom five human antibody Fab libraries, and randomly mutating fourputative solvent-accessible residues in CDR1 (FIG. 23).

A VH, m36, was selected from this library as the highest affinity binderby using the sequential antigen panning (SAP) methodology (Zhang et al.,2003) with HIV-1 Envs from clade B: a truncated Env lacking thetransmembrane portion and the cytoplasmic tail from R2 (gp140_(R2))(Quinnan et al., 1999) or from JRFL (gp140_(JRFL)), and gp120 from Balin complex with CD4 as a fusion protein (gp120_(Bal)-CD4) (Fouts T R etal., 2000). M36 is monomeric in PBS at pH 7.4 as determined by sizeexclusion chromatography, and runs on SDS-PAGE gels and size exclusionchromatography with an apparent molecular weight (MW) of 14-15 kDa whichis close to the calculated MW (14.972 kDa, including the His and FLAGtags) (data not shown). It is highly soluble, thermally stable, and isexpressed at high levels in bacteria (˜30 mg per L of culture) (data notshown). The m36 framework and CDR1 are closest to those encoded by theVH3-23 germline gene; the CDR2—to the VH4-34 (FIG. 24). All m36 CDRscontain negatively charged and neutral but not basic residues. The CDR3sequence is relatively short.

Potent Cross-reactive Neutralization of Pseudotyped HIV-1 Isolates bym36

To determine the potency and breadth of HIV-1 neutralization by m36,viruses pseudotyped with Envs from HIV-1 isolates representing clades A,B, C, D and E were used. M36 neutralized six isolates from clade B, oneisolate from clade C, and one isolate from clade A with potency onaverage two-fold higher (two-fold lower IC₅₀s on molar basis) than thatof the broadly cross-reactive neutralizing CD4i antibody scFv m9 (Zhanget al., 2004) (FIG. 25); m9 is an in vitro matured derivative of X5 andexhibits superior neutralizing activity compared to known cross-reactiveHIV-1 neutralizing antibodies (b12, 4E10, 2F5, 2G12, X5) when testedagainst more than 100 isolates. M36 exhibited remarkable activityagainst the clade C isolate GXC-44 and the clade B isolate NL4-3 withvery low IC₉₀s (FIG. 32). It exhibited lower neutralization activityagainst the clade B isolate 89.6 and the clade-D isolate Z2Z6 comparedto scFv m9. M36 and scFv m9 did not neutralize the clade E isolate GXEat concentrations up to 667 nM. M36 was also on average more potent thanthe peptide C34 (FIG. 25); C34 (10) is a gp41-derived peptide whichexhibits HIV-1 entry inhibitory activity comparable to or higher thanthat of the FDA approved peptide entry inhibitor T20 (DP178, FUZEON™)which shares significant sequence homology with C34 although inhibitsentry by somewhat different mechanism involving binding to multiplesites (Liu S et al., 2005). The inhibitory activity of m36 wasdose-dependent (FIG. 26A). Complete (100%) inhibition of 4 out of 7clade B isolates and the isolate from clade C was achieved at 667 nMconcentration; note that at the equivalent molar concentration C34 didnot completely inhibit any of the isolates tested, and m9 completelyinhibited 3 out of 7 clade B isolates (FIG. 26B). These results suggestthat m36 is a potent cross-reactive neutralizing antibody with potencyand breadth of neutralization for this panel of isolates on averagebetter than that of scFv m9 and C34. These three inhibitors exhibitdifferential neutralization profiles and could be used in combination.

M36 Binding to gp120 is Enhanced by CD4 and Decreased by the CD4-bindingSite Antibody m14

To approximately localize the m36 epitope and begin to elucidate theunderlying mechanisms of neutralization, we measured binding of m36 toEnvs from different isolates alone and in complex with CD4 as well asthe m36 competition with well characterized antibodies. M36 bound togp120_(Bal)-CD4 with high affinity (EC₅₀˜2.5 nM) but not to gp120_(Bal)or to soluble CD4 (sCD4) and BSA, as measured by an ELISA assay (FIG.3A). It also bound to gp140 from a clade C isolate GXC-44(gp140_(GXC-44)) in the presence of sCD4 but notably it did bindalthough weaker to gp140_(GXC-44) alone too (data not shown) in contrastto gp120_(Bal) (data not shown). Similarly, it also bound to anotherclade B Env, a tethered gp140 from 89.6, without complexation with CD4but its binding was enhanced after gp140 bound to CD4 (data not shown).In both cases the binding as function on concentration deviated from aclassical Langmuir-type isotherm likely due to a more complexmulti-stage mechanism of antibody-antigen interactions. As expected fora CD4-induced (CD4i) antibody, m36 competed for binding togp120_(Bal)-CD4 with the CD4i antibody m16 (FIG. 3B) but not with theCD4 binding site (CD4bs) antibody m14 which was used as a negativecontrol. Notably, it also competed for binding to gp140_(GXC-44) in theabsence of CD4 with m14 (data not shown). These results suggest that m36is a cross-reactive CD4i antibody which binds to an epitope localizedclose to the CD4 binding site.

The m36 Epitope is Sterically Restricted

In an attempt to further elucidate possible mechanisms ofneutralization, access to the m36 epitope was examined to determinewhether it was sterically restricted. To answer this question severalm36 fusion proteins with MWs ranging from 18 to 115 kDa (FIG. 28 andFIG. 29) were designed and tested. First, the binding of the m36 fusionproteins to Env complexed with CD4 was carried out to assure that theadditional protein does not interfere with binding. Next, theneutralizing activity of the fusion proteins was evaluated.

The m36 was fused to a serum albumin binding peptide (SAbp), human IgG1CH3 domain and Fc without a peptide linker; these fusion proteins,m36SAbp (MW˜18 kDa), m36CH3 (MW˜60 kDa) and m36b0Fc (MW˜80 kDa),respectively (FIG. 4), were expressed in E. coli HB2151 and purified. Inthe other three fusion proteins m36 was joined with human IgG1 Fc by ahuman IgG1 hinge (m36h1Fc, MW˜110 kDa), a camel IgG2 hinge (m36c2Fc,MW˜115 kDa) or a human IgG3 hinge (m36h3Fc, MW˜115 kDa), respectively(FIG. 28), and expressed in mammalian 293 suspension cells. All fusionproteins, except m36SAbp, were dimeric in PBS, pH 7.4 as shown onnon-reducing SDS-PAGE gels (FIG. 30). They all exhibited comparable toor higher than m36 binding to gp120_(Bal)-CD4 as measured by ELISA.M36SAbp and m36CH3 showed binding comparable to that of m36 (FIG. 31A);m36b0Fc bound slightly better. The three fusion proteins expressed inmammalian cells (m36h1Fc, m36c2Fc and m36h3Fc) exhibited the highestbinding strengths with EC₅₀˜0.5 nM (FIG. 31B). All antibodies atconcentrations up to 870 nM did not show significant binding togp120_(Bal) in the absence of CD4.

In spite of the preserved or even higher affinity (avidity), all fusionproteins, except m36SAbp, exhibited significantly weaker neutralizingactivity when compared to m36 side by side in the same experiment (FIG.25 and FIG. 32). The increased size of m36CH3 resulted in loss ofneutralization against 7 of 10 isolates compared to m36, and for thoseneutralized (IIIB, 89.6 and NL4-3) the IC₅₀s were significantly higherthan the corresponding ones for m36. With an additional increase inmolecular size, m36b0Fc further lost neutralization against the T cellline adapted (TCLA) isolate, IIIB, and had a decreased inhibitoryactivity against 89.6 and NL4-3. Notably, the three fusion proteins withlong flexible linkers neutralized HIV-1 significantly better than thebacterially expressed m36b0Fc which does not have a linker and m36CH3which has much smaller molecular size (FIG. 25 and FIG. 32). Theyneutralized five isolates, one of them (89.6) even with potency higherthan that of m36 likely due to their bivalency leading to avidity andother effects. These isolates were neutralized equally well by m36h1Fc,m36c2Fc and m36h3Fc indicating that further increase in the length ofthe linker may not affect the neutralization activity. Fusion of m36with the relatively much smaller SAbp resulted only in slight but notsignificant decrease of the neutralizing activity (FIG. 25).

Next, it was investigated whether the access to the m36 epitope onintact virions before entry into cells could be enhanced by CD4. M36 andits fusion proteins were preincubated with pseudovirus and in thepresence of a low concentration of sCD4. The m36h1Fc fusion proteinalone at up to 870 nM and sCD4 alone at 8 nM or combined with a controldomain antibody-Fc fusion protein exhibited low neutralizing activity ofabout 25% and 10%, respectively (FIG. 33). Preincubation of the Balpseudovirus with both m36h1Fc and sCD4 resulted in a dramatic increasein neutralization—up to 100% (FIG. 33); similar increase was alsoobserved with JRFL pseudovirus. Taken together these results suggestthat the m36 epitope is sterically obstructed and fully accessibleduring virus entry only by relatively small size molecules.

The results of Example 3 are further discussed as follows.

Discussion:

Example 3 demonstrates that dAbs could bind to conserved structures thatare inaccessible or partially accessible during virus entry formolecules of larger size comparable to that of full-size antibodiesgenerated by the human immune system. Such antibodies could not only bepotentially useful as candidate therapeutics against viruses, includingHIV-1, which can protect highly conserved structures that are vital forvirus replication, but can also help identify those conserved structureswith implications for the development of small molecule inhibitors, andelucidation of mechanisms of entry and evasion of immune responses.

The binding of the Env to receptor and coreceptor molecules may resultin the exposure of conserved structures that could be used as antigensfor selection of cross-reactive neutralizing antibodies. Theidentification and characterization of the potent broadly cross-reactivehuman Fab X5 (Moulard et al., 2002) provided supporting evidence. Thecrystal structure of its complex with gp120-CD4 enabled the localizationof the highly conserved epitope overlapping the putative coreceptorbinding site very close to the CD4 binding site (Huang et al, 2005).However, unexpectedly, IgG1 X5 on average exhibited lower potency thanFab and scFv likely due to its larger size. Because the crystalstructure of Fab X5 complexed with gp120-CD4 suggested that only itsheavy chain contacts gp120, decreasing the size to a single VH couldfurther increase the potency of X5. However, efforts to isolate stableVH X5 or VH X5-like dAbs by rational design, mutagenesis and screeninghave failed. Similarly, efforts to develop a stable highly soluble VHdAb based on an HIV-1 gp120-specific heavy chain only antibody have alsofailed likely due to certain extent of hydrophobicity that is importantfor its structural stability.

As disclosed in the present application, including in the aboveExamples, the present inventors identified another heavy chain onlyantibody which as an isolated VH, m0, exhibited high stability andsolubility. As outlined in the present application, including in Example2, a large highly diversified library using as a scaffold m0 wasconstructed. The high diversification of the library was achieved by twostrategies—grafting highly diverse CDR2s and CDR3s from five separatelibraries including one from HIV-1-infected individuals, and randomlymutating four residues in the CDR1 to residues frequently found inantibody CDRs. This library was used for selection of m36 and could alsobe useful for isolation of dAbs against other antigens.

Access of full-size antibodies to CD4i epitopes can be restricted duringvirus entry into cells. The crystal structures of two CD4i antibodies,X5 and 17b, in complex with gp120 and sCD4, indicate that access totheir epitopes requires long protruding heavy chain CDR3s. Most of theknown CD4i antibodies have long CDR3s that could play an important rolein accessing sterically restricted areas. Their CDR3s are highly acidic(FIG. 24) and the closest germline VH gene for this group of antibodiesis VH1-69. A smaller group of CD4i antibodies have relatively shortCDR3s, acidic CDR2s and VH1-24 gene usage. M36 appears to be the onlyrepresentative of a third group characterized with short CDR3, acidicCDR1 and VH3-23 gene usage (FIG. 24). Because of its small size it maynot need a long CDR3 for access to sterically restricted structures.

M36 exhibited on average higher neutralizing activity than scFv m9,which has been recently shown to be superior to the best characterizedcross-reactive HIV-1 neutralizing antibodies b12, 2G12, 2F5 and 4E10,and than C34, a peptide similar to the fusion inhibitor T20 (FUZEON™)which is in clinical use. It was hypothesized that a dimer of m36 couldhave even higher potency due to avidity effects and used CH3 as adimerization domain. However, m36CH3 was significantly weaker than m36indicating that the m36 epitope is fully accessible during virus entryonly by antibody domains or smaller molecules (FIG. 25). A larger fusionprotein, M36b0Fc, was poorer inhibitor most likely due to the increasedmolecular size. However, a fusion protein with a human IgG1 hinge regionas a linker between m36 and Fc, m36h1Fc, neutralized better severalisolates than m36CH3 and m36b0Fc despite an increase in size (FIG. 25).For some isolates (89.6, NL4-3 and GXC-44) its potency was as high asthat of m36. It was further hypothesized that the long linker mayprovide a flexibility needed for the m36 to reach its epitope combinedwith an increased binding due to avidity effects resulting from them36h1Fc bivalency. However, fusion proteins with even longer hingeregions (from camel antibodies, m36cFc, or from human IgG3, m36h3Fc) didnot exhibit higher potency than m36h1Fc (FIG. 25) possibly due tocompensation of the flexibility effect by an increase in the effectivehydrodynamic size of the molecules leading to a decrease in theaccessibility of the m36 epitope.

The neutralizing activity of the m36 fusion proteins was dramaticallyincreased for viruses sensitized (pre-triggered) by sCD4 (soluble CD4)to expose the m36 epitope (FIG. 33). These data not only provideevidence for the restricted nature of the m36 epitope during entry butalso suggest the possibility to develop novel m36-based potentialtherapeutics, e.g. fusion proteins of m36 with sCD4 or small moleculemimics of CD4. These molecules could neutralize the virus before itbinds to cell surface-associated CD4 while m36 is likely to exert itsmajor neutralizing activity after the virus binds to the cellsurface-associated CD4 which triggers the conformational changes ingp120 leading to exposure of its epitope.

To our knowledge m36 is the first identified and characterized HIV-1neutralizing human dAb. It could have potential as a therapeutic addinga new target to the growing family of entry inhibitors. Although itshalf-life in vivo is likely to be very short our findings that a fusionprotein with an SAbp (serum albumin-binding peptide) retains about thesame neutralizing activity as m36 indicates the possibility to improveits pharmacokinetics. We found that the m36SAbp binds to serum albuminsfrom human (HSA), bovine (BSA) and mouse (MSA) (data not shown)indicating that such possibility is realistic. The epitope of m36 issterically restricted and may not be directly used to develop potentialvaccine immunogens. However, it is highly conserved and therefore couldbe also useful as a tool to explore mechanisms of entry and tounderstand how HIV-1 guards its conserved structures and evadeneutralizing immune responses.

Having thus described in detail preferred embodiments of the presentinvention, it is to be understood that the invention defined by theabove paragraphs is not to be limited to particular details set forth inthe above description as many apparent variations thereof are possiblewithout departing from the spirit or scope of the present invention.

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What is claimed is:
 1. An isolated domain antibody (i) consisting of theamino acid sequence of SEQ ID NO: 96 (m36), or (ii) having an amino acidsequence with at least 90% sequence identity to framework amino acidsequence SEQ ID NO:4 (m0), and including (a) a CDR1 having anphenylalanine residue at position 28 and 30(Kabat numbering system) andone of alanine, aspartate, serine or tyrosine at positions 27, 29, 31and 32, and (b) at least one of CDR2 or CDR3 of SEQ ID NO:96 (m36),wherein the domain antibody binds an epitope on gp120 with adissociation constant (K_(d)) of from about 1 nm to about 500 nM, theexposure of which is induced by binding of gp120 to CD4.
 2. The domainantibody of claim 1, wherein the domain antibody or fragment isimmunoconjugated to one or more cytotoxic agents, chemotherapeuticagents, natural or synthetic toxins, radioactive isotopes, or antiviralagents.
 3. The domain antibody of claim 2, wherein the antiviral agentis zidovudine, acyclovir, gangcyclovir, vidarabine, idoxuridine,trifluridine, and ribavirin, as well as foscarnet, amantadine,rimantadine, saquinavir, indinavir, amprenavir, lopinavir, ritonavir,adefovir, clevadine, entecavir, or pleconaril.
 4. A pharmaceuticalcomposition comprising a therapeutically effective amount of a domainantibody in accordance with claim 1 for treating HIV-1 infection.
 5. Acomposition comprising an effective amount of a domain antibody orfragment thereof consisting of the amino acid sequence of SEQ ID NO: 96(M36), and a pharmaceutically acceptable carrier.